Gbs toxin receptor

ABSTRACT

A novel GBS toxin receptor, and methods for its preparation and use are provided. GBS toxin receptor polynucleotides and polypeptides are provided as well as detection, screening, and therapeutic methods and pharmaceutical compositions involving such polynucleotides and polypeptides.

TECHNICAL FIELD

This invention provides compositions and methods relating to GBS toxinreceptor polynucleotides and polypeptides. The invention relates to areceptor for a polysaccharide isolated from a bacterial source.

BACKGROUND

Group B β-hemolytic Streptococci (GBS) are ubiquitous microorganisms.GBS is not known to cause any harmful infections in humans except forvery young babies. GBS pneumonia, also called “early-onset disease”, isassociated with high morbidity and mortality in newborn infants.

In a series of studies conducted by Dr. Carl G. Hellerqvist and hisassociates at the Vanderbilt University School of Medicine, Nashville,Tenn., a polysaccharide GBS toxin was identified. This toxin wasdetermined to be a major factor in the complications of GBS pneumonia,and was found to be useful as a therapeutic agent in combating tumorsthough inhibition of vascularization (U.S. Pat. No. 5,010,062).

In addition, as described in U.S. Pat. No. 5,858,991 and WO98/32453, GBStoxin facilitates wound healing in patients by minimizing scarring andaccelerating healing, and reduces wound-related tumor progression.

WO98/32452 and WO98/32448 describe the use of GBS toxin as a therapeuticagent for treating patients with chronic inflammatory diseases, such asrheumatoid arthritis and psoriasis, and for enhancing repair of neuralinjury.

Prior to this invention, receptors for GBS toxin had not beenidentified. The inventors, believing receptors of GBS toxin to reside oncells in the developing vasculature of tissues undergoing angiogenesisin the conditions described above, embarked upon a series of experimentsresulting in the present invention.

SUMMARY OF THE INVENTION

For the first time, novel receptors for group B β-hemolyticStreptococcus GBS toxin (GBS toxin receptor) have been identified. Oneaspect of the invention provides a polypeptide comprising a GBS toxinreceptor or polypeptide fragment thereof. Preferred embodiments includemammalian GBS toxin receptors. Also provided is an antibody thatrecognizes GBS toxin receptor or a fragment thereof. The polypeptide ofthe invention can be used, inter alia, for the screening of compoundsthat can be used to treat or prevent conditions arising from pathologicor hypoxia-driven angiogenesis or neovascularization, such as, forexample, cancerous tumors, chronic inflammatory disease, scarring duringwound healing, keloids, neural injury, and reperfusion injury.

Another aspect of the invention provides a polynucleotide encoding a GBStoxin receptor or a fragment thereof and a polynucleotide hybridizableto such polynucleotide. Preferred polynucleotides are at least 10 basesin length and comprise a nucleic acid sequence encoding, or arecomplementary to a nucleic acid sequence encoding, a mammalian GBS toxinreceptor or a polypeptide fragment thereof.

A third aspect of the invention is a complex comprising a GBS toxinbound to a mammalian toxin receptor or fragment thereof. Also providedis a method of forming such complex. The method comprises contacting aGBS toxin with a polypeptide comprising a mammalian GBS toxin receptor,or fragment thereof that can bind GBS toxin, under conditions thatpermit specific binding of the GBS toxin to the polypeptide, andallowing the complex to form.

Yet another aspect of the invention is a method for purifying a compoundthat binds a GBS toxin receptor. The method comprises providing apolypeptide comprising a mammalian GBS toxin receptor, or fragmentthereof that binds GBS toxin, contacting the polypeptide with a samplecomprising the compound under conditions that allow specific binding ofthe compound to the polypeptide, and separating the bound compound fromthe remainder of the sample.

Another aspect of the invention is a method of determining the presenceor absence of GBS toxin in a sample. The method comprises contacting thesample with a polypeptide comprising a mammalian GBS toxin receptor, orfragment thereof that binds GBS toxin, under conditions that allowspecific binding of GBS toxin to the GBS toxin receptor, and determiningwhether specific binding of GBS toxin has occurred. Presence of GBStoxin in a sample obtained from a neonate is indicative of early onsetdisease.

A sixth aspect of the invention is a method for detecting pathologicvasculature in a mammalian tissue. The method comprises detecting thepresence of a GBS toxin receptor. The method can be used for detectingor monitoring a variety of medical conditions associated withangiogenesis or neovascularization, such as, for example, detectingmetastasis of a cancerous tumor, or monitoring the margin of a tumor ina mammal undergoing a therapy for cancer.

Another aspect of the invention provides methods for the identificationof drug candidates for the treatment of medical conditions characterizedby pathologic and/or hypoxia-driven angiogenesis or neovascularization.One embodiment is a method for identifying a compound that specificallybinds a mammalian GBS toxin receptor. The method comprises combining atest compound with a mammalian GBS toxin receptor, or fragment thereofthat can bind GBS toxin, under conditions that allow specific binding tooccur, and detecting a complex formed between the test compound and thepolypeptide. Another embodiment is a method for determining cytotoxicityof a test chimeric compound. The method comprises exposing a cellexpressing a mammalian GBS toxin receptor, or fragment thereof thatbinds GBS toxin, to a test chimeric compound comprising a cytotoxicagent coupled to GBS toxin, and detecting signs of toxicity. Yet anotherembodiment is a method for identifying an inhibitor of a GBS toxinreceptor by incubating test cells that express GBS toxin receptor, or afragment thereof, in the presence and absence of a test compound andunder conditions in which the cells incubated in the absence of the testcompound can proliferate or migrate, and comparing the proliferation ormigration of the test cells incubated in the presence and absence of thetest compound, wherein less proliferation or migration in the presenceof the test compound is indicative of the test compound being aninhibitor of the GBS toxin receptor. An inhibitor of endothelial cellproliferation or migration can be identified by the above method,wherein less proliferation or migration of test cells in the presence ofthe test compound is indicative of the test compound being an inhibitorof endothelial cell proliferation or migration. A therapeutic compoundfor the treatment or prevention of a medical condition characterized bypathologic angiogenesis or neovascularization can also be identified bythe above method, wherein less proliferation or migration of test cellsin the presence of the test compound is indicative of the test compoundbeing a candidate therapeutic compound for the treatment or preventionof the medical condition.

The invention also provides a method for identifying a compound whichinhibits binding of a GBS toxin to a mammalian GBS toxin receptor. Themethod comprises simulating and selecting the most probableconformations of a mammalian GBS toxin receptor, designing a chemicallymodified analog that substantially mimics the energetically mostprobable three-dimensional structure of the polypeptide, chemicallysynthesizing the analog, and evaluating the bioactivity of the analog.Also provided is a method for identifying a compound which binds to amammalian GBS toxin receptor. The method comprises simulating andselecting the most probable conformations of a mammalian GBS toxinreceptor, deducing the most probable binding domains of the polypeptide,designing a compound that would form the energetically most probablecomplexes with the polypeptide, chemically synthesizing the compound,and evaluating the bioactivity of the compound.

Another aspect of the invention is a method for the prevention ortreatment of neonatal onset disease in a human neonate by administeringan inhibitor of binding of GBS toxin to a human GBS toxin receptor.

Yet another aspect of the invention is a method for inhibitingpathologic or hypoxia-driven endothelial cell proliferation or migrationin a mammalian tissue. The method comprises specifically binding amolecule to a GBS toxin receptor present on the surface of at least onecell in the tissue, the molecule being selected from the groupconsisting of a compound that can evoke an inflammatory response whenbound to a GBS toxin receptor in a mammal, a chimeric compoundcomprising a cytotoxic compound coupled to a compound that specificallybinds the GBS toxin receptor, an inhibitor of GBS toxin receptorphosphorylation, and an inhibitor of GBS toxin receptor activity.

The invention also provides a GBS toxin receptor or fragment thereof, aninhibitor of a GBS toxin receptor, or an inhibitor of binding of a GBStoxin to a GBS toxin receptor, for use in a method of treatment of thehuman or animal body or for the manufacture of a medicament for thetreatment of a medical condition characterized by pathologic orhypoxia-driven angiogenesis or neovascularization. Also provided is achimeric compound comprising a cytotoxic agent coupled to a compoundthat binds GBS toxin receptor for use in a method of treatment of thehuman or animal body.

Also provided are pharmaceutical compositions comprising an inhibitor ofa GBS toxin receptor and/or a chimeric compound comprising a cytotoxicagent coupled to a compound that binds GBS toxin receptor, and apharmaceutically acceptable carrier.

The invention also provides kits comprising a GBS toxin receptor orfragment and/or reagents for detecting the presence of a GBS toxinreceptor or polypeptide fragment thereof or the presence of apolynucleotide encoding same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a process of rational drug design.

FIGS. 2A and 2B depict the results of immunohistochemical analysis ofGBS toxin receptor expression in cancerous and normal human ovarytissue, respectively, using antibody Pab55 as described in Example 4.

FIGS. 3A and 3B depict the results of immunohistochemical analysis ofGBS toxin receptor expression in cancerous and normal human ovarytissue, respectively, using antibody Pab57 as described in Example 4.

FIGS. 4A-4C depict the targeted delivery of a chimeric compound to GBStoxin receptor expressed in a cancerous tissue as described in Example6.

DESCRIPTION OF SPECIFIC EMBODIMENTS DEFINITIONS

Generally, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. The nomenclature used herein and thelaboratory procedures in cell culture, molecular genetics, and nucleicacid chemistry and hybridization described below are those well knownand commonly employed in the art. Standard techniques are used forrecombinant nucleic acid methods, polynucleotide synthesis, andmicrobial culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification steps supplied bymanufacturers are typically performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (See generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.) which are provided throughout this document.The nomenclature used herein and the laboratory procedures in analyticalchemistry, organic synthetic chemistry, and pharmaceutical formulationdescribed below are those well known and commonly employed in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical formulation and delivery, and treatment ofpatients. As employed throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings:

By “GBS toxin receptor” is meant a proteinaceous molecule capable ofbinding a toxin from Group B β-hemolytic Streptococcus bacteria (GBStoxin), such as, for example, CM101. A GBS toxin receptor is usuallyfound in nature on the surface of a cell. Recombinant membrane bound andsoluble GBS toxin receptors can be produced by laboratory techniquesknown in the art and described herein.

The term “isolated polynucleotide” referred to herein means apolynucleotide that has been subjected to manipulation, such that theisolated polynucleotide is no longer associated with the chromosome orcell that the polynucleotide is normally associated with in nature inthe same manner as it is normally associated in nature. An example of an“isolated polynucleotide” is a polynucleotide of genomic, recombinant,or synthetic origin or some combination thereof.

The term “isolated protein” referred to herein means a protein that isno longer associated with the cell that the protein is normallyassociated with in nature in the same manner as it is normallyassociated in nature, such as (1) a protein free of at least some otherproteins from the same source, (2) a protein expressed by a cell from adifferent species, (3) a protein that does not occur in nature, and (4)a protein produced from cDNA, recombinant RNA, or synthetic origin orsome combination thereof.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus.

The term “naturally occurring” means found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) found in nature and which has not been intentionallymodified by man in the laboratory is naturally-occurring.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is necessary for expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.The term includes single- and double-stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring and non-naturally occurring oligonucleotide linkages.

An oligonucleotide is usually a polynucleotide 200 bases or fewer inlength. Preferably oligonucleotides are minimally 10 to 60 bases inlength and most preferably 15-35 bases in minimal length.Oligonucleotides are usually single-stranded, e.g. for probes; althougholigonucleotides may be double-stranded, e.g. for use in theconstruction of a gene mutant. Oligonucleotides of the invention can beeither sense or antisense oligonucleotides. The term “naturallyoccurring nucleotides” referred to herein includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” referred to hereinincludes nucleotides with modified or substituted sugar groups and thelike. The term “oligonucleotide linkages” referred to herein includesoligonucleotides linkages such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phoshoraniladate, phosphoroamidate, and the like. An oligonucleotide caninclude a label for detection, if desired.

By “complementary” or “complement” is meant that wherever adenineappears in a first nucleic acid sequence, thymine or uracil is found inthe “complementary” sequence and vice versa, and wherever guanineappears in a first nucleic acid sequence, cytosine is found in the“complementary” sequence and vice versa.

The term “sequence identity” describes the proportion of base matchesbetween two nucleic acid sequences or the proportion of amino acidmatches between two amino acid sequences, i.e. the degree of identitybetween two sequences. When sequence identity is expressed as apercentage, e.g., 50%, the percentage denotes the proportion of exactmatches over the length of sequence from a GBS toxin receptor sequencethat is compared to some other sequence. Various computer alignmentprograms can be used to determine sequence identity. In its simplestform, % identity is calculated by dividing the number of exact matchesbetween two nucleic acid sequences or between two amino acid sequencesby the total number of nucleotides or amino acids in the referencesequence. For example, if there are 300 matches between sequences 400amino acids in length, the sequences have 75% identity. Uracil andthymine are considered identical when comparing a ribonucleic acidsequence with a deoxyribonucleic acid sequence.

As applied to polynucleotides, the term “substantial identity” meansthat two nucleic acid sequences when optimally aligned, such as by theprogram BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), shareat least about 85%, preferably at least about 90% sequence identity andmost preferably 95% or greater sequence identity. When using computeralignment programs, gaps (in either of the two sequences) are permittedto maximize matching; gap lengths of 15 bases or less are usually used;6 bases or less are preferred; 2 bases or less are most preferred. Whenusing oligonucleotides as probes or in treatments, the sequence identitybetween the target nucleic acid and the oligonucleotide sequence isgenerally not less than 17 target base matches out of 20 possibleoligonucleotide base pair matches (85%); preferably not less than 9matches out of 10 possible base pair matches (90%), and most preferablynot less than 19 matches out of 20 possible base pair matches (95%).

Preferably, bases which are not identical nevertheless are part of adegenerate codon that encodes the same amino acid at that amino acidposition. Alternatively, bases which are not identical preferably arepart of a degenerate codon that encodes a conservative amino acidsubstitution for that amino acid position.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned by the BLAST computerprogram, share at least about 80 percent sequence identity, preferablyat least about 86 percent sequence identity, more preferably at leastabout 95 percent sequence identity, even more preferably at least about99 percent sequence identity up to having one amino acid difference, andmost preferably share 100% identity. Gaps (in either of the twosequences being matched) are allowed in maximizing matching; gap lengthsof 5 or less are preferred with 2 or less being more preferred.Preferably, residue positions which are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acid substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

The term “hybridizable under high stringency conditions” referred toherein means capable of specific binding under conditions whereby onlynucleic acid sequences having a substantial identity of greater than 95%with respect to each other will hybridize. These conditions are known inthe art and discussed herein.

The term “degenerate codon” means any of the nucleotide codon tripletsencoding a desired amino acid according to the genetic code. Codons canbe selected based upon known preferred codon usage in a host organismsuch as E. coli.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length DNA sequence. Fragments typically are at least 3 aminoacids long, preferably are 5-10 amino acids long, more preferably are10-50 amino acids long, even more preferably are more than 50 aminoacids long and comprise at least one extracellular domain of a GBS toxinreceptor. Most preferred are fragments that comprise the entireextracellular domains of a GBS toxin receptor, and preferably alsocomprise portions of transmembrane and intracellular domains sufficientto maintain the polypeptide fragment in a functional stereochemicalconformation on the surface of a cell, lipid membrane, liposome,micelle, or other lipophilic structure.

The term “immunologically reactive” means having antigenic properties orbeing capable of being specifically bound by an antibody that canspecifically bind GBS toxin receptor. A substance has antigenicproperties if it can generate monoclonal or polyclonal antibodies whenadministered to an animal under conditions known in the art tofacilitate the production of antibodies that will recognize and bind aparticular antigen.

A “heterologous polypeptide” is a polypeptide different frompolypeptides normally produced by a particular cell. For example, a GBStoxin receptor polypeptide or fragment thereof that is producedrecombinantly in a cell that does not normally produce such GBS toxinreceptor polypeptide or fragment thereof, is a heterologous polypeptide.A second polypeptide joined to a GBS toxin receptor polypeptide orfragment thereof is also a heterologous polypeptide if it is not joinedto a GBS toxin receptor polypeptide in nature.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcolorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I), fluorescent labels(e.g., rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), chemiluminescent, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In some embodiments, labels are attachedby spacer arms of various lengths to reduce potential steric hindrance.

The term “compound” as used herein preferably refers to a peptidic,peptidomimetic, organic, or other chemical molecule and also refers to anucleic acid molecule or chemical derivative thereof. The compound caninteract with, or be, the polynucleotides or polypeptides of theinvention.

The singular forms “a”, “an” and “the” include plural referents unlessthe context clearly dictates otherwise.

The SEQ ID NOs of the nucleic acid and amino acid sequences describedherein are summarized below in Table 1.

TABLE 1 Nucleic Acid and Amino Acid Sequences SEQ ID NO: Type ofSequence Description SEQ ID NO: 1 nucleic acid Partial human GBS toxinreceptor (HP55) SEQ ID NO: 2 amino acid Partial human GBS toxin receptor(HP55) SEQ ID NO: 3 nucleic acid Sheep GBS toxin receptor (SP55) SEQ IDNO: 4 amino acid Sheep GBS toxin receptor (SP55) SEQ ID NO: 5 nucleicacid Primer SEQ ID NO: 6 nucleic acid Primer SEQ ID NO: 7 nucleic acidFull-length human GBS toxin receptor (HP59) SEQ ID NO: 8 amino acidFull-length human GBS toxin receptor (HP59) SEQ ID NO: 9 nucleic acidHuman/Sheep consensus GBS toxin receptor coding region (with base codesa, c, g, t, m, r, w, s, y, k) SEQ ID NO: 10 amino acid Human/Sheepconsensus GBS toxin receptor coding region (translation of SEQ ID No: 9)SEQ ID NO: 11 nucleic acid Human/Sheep consensus GBS toxin receptorcoding region (with base codes a, c, g, t, n) SEQ ID NO: 12 amino acidHuman/sheep consensus GBS toxin receptor coding region (translation ofSEQ ID NO: 11)

The headings provided herein describe the general topic discussed andare not intended to be exclusive of information discussed in othersections. Frequently, information, methods, compositions, and otheraspects may be applicable to more than one embodiment of the inventionand can be so combined.

Introduction

GBS toxin binds to tissues undergoing pathologic, hypoxia-driven, andembryologic angiogenesis or neovascularization. The inventors haveidentified at least two mammalian GBS toxin receptors, which aredescribed herein. Examples 1 and 2 describe the cloning andcharacterization of some GBS toxin receptors. The inventors haveclassified GBS toxin receptor as an integral protein with seventransmembrane domains. The predicted segments are shown in Table 7. Theprotein has several putative sites for phosphorylation by cAMP-dependentkinase, protein kinase C (PKC), and casein kinase II (CK2). Typically,such integral proteins, upon binding of a molecule (e.g., a ligand or anextracellular messenger), undergo a conformational change whichfacilitates phosphorylation at phosphorylation sites such as thosediscussed above. The phosphorylation of the protein at these sites maytrigger a signal transduction cascade, which often results inproliferation or other nuclear responses of the cells which have beenexposed to the binding molecule. Angiogenesis or neovascularizationinvolves proliferation and migration of endothelial cells. As discussedin greater detail in Examples 4 and 5, GBS toxin receptor expression iscorrelated with medical conditions involving pathologic, hypoxia-driven,and embryogenic angiogenesis or neovascularization. GBS toxin receptorpolypeptides can be used for a variety of purposes, including screeningfor compounds that can inhibit endothelial cell proliferation and/ormigration mediated by GBS toxin receptor and screening for cytotoxicchimeric compounds that can bind to and destroy cells expressing GBStoxin receptor. GBS toxin receptor polynucleotides can be used for avariety of purposes, including the design of antisense polynucleotidesthat can block translation of messenger RNA encoding GBS toxin receptor.

Polynucleotides

One aspect of the invention provides for isolated polynucleotides atleast ten bases in length encoding or complementary to a nucleic acidsequence encoding a GBS toxin receptor or a fragment derived therefrom.Preferably, the GBS toxin receptor is a mammalian GBS toxin receptor,more preferably an ovine, bovine or feline GBS toxin receptor, and mostpreferably a human GBS toxin receptor. The isolated polynucleotides canbe naturally occurring or non-naturally occurring. The isolatedpolynucleotides can comprise a DNA sequence or an RNA sequence in whichevery T is replaced with U. For purposes of determining percentageidentity, T is considered equivalent to U. Preferably, thepolynucleotides include alleles of an ovine, bovine, feline or human GBStoxin receptor, and can include alleles of GBS toxin receptor of othermammals. These polynucleotides can be isolated using polynucleotidesderived from SEQ ID NOs: 1, 3, 7, 9 and 11, as described further below.

Polynucleotides, oligonucleotides and fragments of the inventionselectively hybridize to nucleic acid strands under hybridization andwash conditions that minimize appreciable amounts of detectable bindingto nonspecific nucleic acids. The polynucleotides can be hybridizableunder high stringency conditions to a nucleic acid molecule having anucleic acid sequence comprising at least 20 contiguous polynucleotides,preferably at least 30 contiguous nucleotides of SEQ ID NO: 1 or SEQ IDNO: 3, and even more preferably to the nucleic acid sequence of SEQ IDNO: 1, 3, 7, 9 or 11 or the complement of SEQ ID NO: 1, 3, 7, 9 or 11.Such polynucleotides can be used for performing selective, highstringency hybridization and are particularly useful for performingamplification of nucleic acid by polymerase chain reaction (PCR) todetermine the presence or absence of GBS toxin receptor in a sample, forisolating a naturally occurring nucleic acid encoding a GBS toxinreceptor (see Example 3), as antisense molecules for blockingtranslation of GBS toxin receptor mRNA. Particularly preferred arepolynucleotides hybridizable under high stringency conditions to anucleic acid molecule having a nucleic acid sequence comprising thenucleic acid sequence of nucleotides 266 to 1870 of SEQ ID NO: 7 (theputative full length coding region of a human GBS toxin receptor,excluding the start codon), nucleotides 266 to 1870 of SEQ ID NO:7 (theputative full length coding region of a human GBS toxin receptor,including the start codon), nucleotides 61 to 1542 of SEQ ID NO:1 (thepartial coding region of a human GBS toxin receptor, excluding the startcodon), nucleotides 58 to 1542 of SEQ ID NO: 1 (the partial codingregion of a human GBS toxin receptor, including the start codon),nucleotides 87 to 1568 of SEQ ID NO: 3 (the coding region of a sheep GBStoxin receptor, excluding the start codon), nucleotides 84 to 1568 ofSEQ ID NO:3 (the coding region of a sheep GBS toxin receptor, includingthe start codon), or a complementary nucleic acid sequence thereof.

The polynucleotides can have an identity to the nucleic acid sequence ofa corresponding region of SEQ ID NO: 1, 3 or 7 or the complement of acorresponding region of SEQ ID NO: 1, 3 or 7 in the range of about 85%to 100%, preferably greater than about 87% identity, more preferablygreater than about 95% identity, and most preferably about 99% to 100%identity. Particularly preferred are polynucleotides comprising thenucleic acid sequence of nucleotides 266 to 1870 of SEQ ID NO: 7, ornucleotides 87 to 1568 of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO:11, or acomplementary nucleic acid sequence thereof.

Preferably, the polynucleotides comprise a nucleic acid sequenceencoding, or complementary to a nucleic acid sequence encoding, apolypeptide having an identity to the amino acid sequence of a fragmentof a GBS toxin receptor in the range of about 85% to 100%, morepreferably greater than 86% identity, even more preferably greater than95% identity, and most preferably 99% to 100% identity. Preferably, thefragment binds GBS toxin. Preferred fragments comprise all or a portionof residues 1 to 495 of SEQ ID NO: 2 or all or a portion of residues 1to 536 of SEQ ID NO: 8. Particularly preferred are polynucleotidescomprising a nucleic acid sequence encoding a polypeptide having 100%identity to the amino acid sequence of residues 1 to 495 of SEQ ID NO:4, residues 1 to 495 of SEQ ID NO: 2, or residues 1 to 536 of SEQ IDNO:8.

Polynucleotides encoding naturally occurring GBS toxin receptor can beisolated from various tissue sources and cell cultures from differentspecies that produce such a receptor by the methods described herein,such as, for example, cells from tumor endothelium, synovial tissue inrheumatoid arthritis, or hypoxic tissue deprived of or restricted fromblood flow, such as in reperfusion injury or wounded tissue. Suchpolynucleotides can be isolated by hybridization using probes or bypolymerase chain reaction using oligonucleotides, as well as byimplementing other molecular biology techniques known in the art. Suchprobes and oligonucleotides typically comprise various regions of thesequence of SEQ ID NO: 1, 3, 7, 9 or 11, preferably of SEQ ID NO: 1, 3,or 7, or encode various regions of the sequence of SEQ ID NO. 2, 4, 8,10or 12, preferably of SEQ NO: 2, 4 or 8.

Polynucleotides useful for cloning genes encoding GBS toxin receptors ofvarious organisms can be determined by comparing the amino acidsequences of homologous proteins. (see Table 4). For example, conservedregions can be targeted for the synthesis of oligonucleotides ordegenerate oligonucleotides to be used as probes for hybridization ornucleic acid amplification, techniques discussed further below and inExample 3. Stringency can be varied to achieve selective hybridizationconditions whereby nucleic acid sequences having less than 95% identitywith respect to each other will hybridize. These conditions are known inthe art and discussed herein and examples are provided. Generally, thenucleic acid sequence identity between the polynucleotides,oligonucleotides, and fragments of the invention and a nucleic acidsequence of interest will be at least about 85%, and more typically withpreferably increasing identities of at least about 90%, 95%, 99%, and100%.

Polynucleotides can be used as probes under high stringency washconditions and with corresponding hybridization conditions, as known inthe art. Small polynucleotides, for example, polynucleotides 200 basesor fewer in length, are often referred to in the art asoligonucleotides. Techniques for using polynucleotides as probes todetect the same or related nucleic acid sequences is well known in theart. See, for example, Sambrook et al, especially Chapter 11, the textof which is herein incorporated by reference. Usually, probes can bemade from polynucleotides that are 10 to 200 bases in length. Preferablyprobes are made from polynucleotides 10 to 60 nucleotides in length andmost preferably 12 to 40 bases in length. Specific probes can bedesigned based on results obtained using nucleic acid homology computerprograms such as FASTA, which uses the method of Pearson and Lipman(Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988)) and shows the degree ofidentity between compared sequences. The size of the probe is dependentupon the region of the gene to which it will be hybridized. The size ofthe probe increases as the degree of homology to undesirable nucleicacid sequences increases. A probe 10-50 nucleotides in length can beused, preferably more than 50 nucleotides, even more preferably morethan 100 nucleotides, and most preferably a probe made from the entirecoding region of a GBS toxin receptor will be used. To decrease thenumber of false positives, preferably two probes are used to identifyclones that bind to both probes under hybridization and wash conditions.Oligonucleotides can be synthesized on an Applied BioSystemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Typically, hybridization and washing conditions are performed ataccording to conventional hybridization procedures. Typicalhybridization conditions for screening plaque lifts (Benton and Davis(1978) Science 196: 180) can be: 50% formamide, 5×SSC (sodium chloride,sodium citrate) or SSPE (sodium chloride, sodium phosphate, EDTA), 1-5×Denhardt's solution, 0.1-1% SDS, 100-200 μg sheared heterologous DNA ortRNA, 0-10% dextran sulfate, 1×10⁵ to 1×10⁷ cpm/ml of denatured probewith a specific activity of about 1×10⁸ cpm/pg, and incubation at 42° C.for about 6-36 hours. Prehybridization conditions are essentiallyidentical except that probe is not included and incubation time istypically reduced. Washing conditions are typically 1-3×SSC, 0.1-1% SDS,42-70° C. with change of wash solution at about 5-30 minutes. Cognatebacterial sequences, including allelic sequences, can be obtained inthis manner. For high stringency hybridization conditions, variousparameters can be altered to increase the stringency of hybridization,such as by increasing the temperature of incubation with the labeledprobe. Preferably, for greater flexibility in experimental design, theprobe can be hybridized at a lower temperature, such as, for example,room temperature and the stringency can then be modified by altering thesalt concentration and temperature of the wash solutions. For highstringency a wash temperature of greater than or equal to 42° C. can beused, such as, for example, 68° C., in a wash buffer having a saltconcentration less than 3×SSC, such as, for example, 0.1×SSC. In somecases, TMACL can also be used, particularly for polynucleotides rich inG-C base pairs in order to decrease non-specific binding. A lowerstringency wash can be used to hybridize polynucleotides with loweridentities or polynucleotides that are less than 60 base pairs inlength. For a low stringency wash, temperatures of less than or equal to42° can be used in a wash buffer having a salt concentration of greaterthan or equal to 2×SSC.

The invention includes methods for amplification of target nucleicacids, such as the polymerase chain reaction (“PCR”) technique. The PCRtechnique can be applied to identify related sequences in the genomes ofvarious organisms and to detect nucleotide sequences in suspectedsamples, using oligonucleotide primers spaced apart from each other andbased on the genetic sequence set forth herein. The primers arecomplementary to opposite strands of a double-stranded DNA molecule andare typically separated by from about 50 to 450 nucleotides or more(usually not more than 2000 nucleotides). This method entails preparingthe specific oligonucleotide primers followed by repeated cycles oftarget DNA denaturation, primer binding, and extension with a DNApolymerase to obtain DNA fragments of the expected length based on theprimer spacing. Extension products generated from one primer serve asadditional target sequences for the other primer. The degree ofamplification of a target sequence is controlled by the number of cyclesthat are performed and is theoretically calculated by the simple formula2n where n is the number of cycles. Given that the average efficiencyper cycle ranges from about 65% to 85%, 25 cycles produce from 0.3 to4.8 million copies of the target sequence. The PCR method is describedin a number of publications, including Saiki et al., Science (1985)230:1350-1354; Saiki et al., Nature (1986) 324:163-166; and Scharf etal., Science (1986) 233:1076-1078. Also see U.S. Pat. Nos. 4,683,194;4,683,195; and 4,683,202, the text of each patent is herein incorporatedby reference. Additional methods for PCR amplification are described in:PCR Technology: Principles and Applications for DNA Amplification ed. HA Erlich, Freeman Press, New York, N.Y. (1992); PCR Protocols: A Guideto Methods and Applications, eds. Innis, Gelfland, Snisky, and White,Academic Press, San Diego, Calif. (1990); Manila et al. (1991) NucleicAcids Res. 19: 4967; Eckert, K. A. and Kunkel, T. A. (1991) PCR Methodsand Applications 1: 17, and; PCR, eds. McPherson, Quirkes, and Taylor,IRL Press, Oxford, all of which are incorporated herein by reference.

In yet another embodiment, an antisense polynucleotide can beadministered to a mammal to treat or prevent a medical conditioninvolving pathologic and/or hypoxia-driven angiogenesis. The antisenseoligonucleotides of the invention can be synthesized by any of the knownchemical oligonucleotide synthesis methods. Such methods are generallydescribed, for example, in Winnacker, From Genes to Clones: Introductionto Gene Technology. VCH Verlagsgesellschaft mbH (H., Ibelgaufts trans.1987). Any of the known methods of oligonucleotide synthesis can beutilized in preparing the instant antisense oligonucleotides. Theantisense oligonucleotides are most advantageously prepared by utilizingany of the commercially available, automated nucleic acid synthesizers.The device utilized to prepare the oligonucleotides described herein,the Applied Biosystems 380B DNA Synthesizer, utilizes—cyanoethylphosphoramidite chemistry. Antisense oligonucleotides hybridizable withany portion of the mRNA transcript can be prepared by theoligonucleotide synthesis methods known to those skilled in the art.While any length oligonucleotide can be utilized in the practice of theinvention, sequences shorter than 12 bases may be less specific inhybridizing to the target GBS toxin receptor mRNA, and may be moreeasily destroyed by enzymatic digestion. Hence, oligonucleotides having12 or more nucleotides are preferred. Sequences longer than 18 to 21nucleotides may be somewhat less effective in inhibiting GBS toxinreceptor translation because of decreased uptake by the target cell.Thus, oligomers of 12-21 nucleotides are most preferred in the practiceof the present invention, particularly oligomers of 12-18 nucleotides.Oligonucleotides complementary to and hybridizable with any portion ofthe GBS toxin receptor mRNA transcript are, in principle, effective forinhibiting translation of the transcript, and capable of inducing theeffects herein described. Translation is most effectively inhibited byblocking the mRNA at a site at or near the initiation codon. Thus,oligonucleotides complementary to the 5′ region of the GBS toxinreceptor mRNA transcript are preferred. Secondary or tertiary structurewhich might interfere with hybridization is minimal in this region.Moreover, sequences that are too distant in the 3′ direction from theinitiation site can be less effective in hybridizing the mRNAtranscripts because of a “read-through” phenomenon whereby the ribosomeis postulated to unravel the antisense/sense duplex to permittranslation of the message. (see, e.g. Shakin, J. Biochemistry 261,16018 (1986)). The antisense oligonucleotide is preferably directed to asite at or near the ATG initiation codon for protein synthesis.Oligonucleotides complementary to a portion of the GBS toxin receptormRNA including the initiation codon are preferred. While antisenseoligomers complementary to the 5′ region of the GBS toxin receptortranscript are preferred, particularly the region including theinitiation codon, it should be appreciated that useful antisenseoligomers are not limited to those complementary to the sequences foundin the translated portion of the mRNA transcript, but also includesoligomers complementary to nucleotide sequences contained in, orextending into, the 5′ and 3′ untranslated regions. Antisensenucleotides or antisense expression constructs can find use to treat orprevent diseases associated with pathologic or hypoxia-drivenangiogenesis and neovascularization, as inappropriate expression of GBStoxin receptor results in hyperproliferation of endothelial cells.

In one embodiment, the polynucleotides of the invention can exist inlinear form. In another embodiment, the polynucleotides can exist incircular form as part of a plasmid.

In yet another embodiment, a probe or PCR primer comprises a group ofpolynucleotide species containing different degenerate codons at variouspositions, which polynucleotides encode, or are complementary tosequences encoding, a GBS toxin receptor in whole or in part. Suchpolynucleotides can be useful for isolating nucleic acid sequencesencoding polypeptides having at least about 85% identity to the aminoacid sequence of sheep or human GBS toxin receptor, such as, forexample, GBS toxin receptors of other organisms. Typically, suchpolynucleotides are synthesized chemically as described above byprogramming a synthesizer to incorporate a particular combination ofnucleic acid residues at a certain position. Typical designations areshown in Table 2.

TABLE 2 Base Codes Symbol Meaning A A; adenine C C; cytosine G G;guanine T T; thymine U U; uracil M A or C R A or G W A or T/U S C or G YC or T/U K G or T/U V A or C or G; not T/U H A or C or T/U; not G D A orG or T/U; not C B C or G or T/U; not A N A or C or G or T/U

Polypeptides

Another aspect of the invention provides polypeptides comprising (1) thefull length GBS toxin receptor protein or a naturally occurring allelicvariant thereof, (2) fragments of at least 3 amino acids of the aminoacid sequence of SEQ ID NO: 2, 4, 8, 10 or 12, and (3) a GBS toxinreceptor protein, polypeptide, or polypeptide fragment having an aminoacid identity in the range of about 80% to 100% to the amino acidsequence of a corresponding region of SEQ ID NO: 2, 4 or 8. Preferredfragments of the amino acid sequence of SEQ ID NO: 2, 4, 8, 10 or 12,are at least 5, 6, 7, 8 or 9 amino acids in length and areimmunologically reactive, i.e., immunogenic. More preferred arefragments at least 25 amino acids in length and fragments comprising theamino acid sequence of residues 181 to 419 of SEQ ID NO: 2 or residues 1to 240 of SEQ ID NO: 4. Most preferred are fragments that can bind GBStoxin. Preferably, the GBS toxin receptor protein, polypeptide, orpolypeptide fragment has an amino acid identity to the amino acidsequence of a corresponding region of SEQ ID NO: 2, 4 or 8 of at leastabout 86%, more preferably at least about 95% identity, even morepreferably at least about 99% identity up to having one amino aciddifference, and most preferably 100% identity. Preferred polypeptideshave at least about 89% identity, more preferably at least about 95%identity, even more preferably at least about 99% identity up to havingone amino acid difference, and most preferably 100% identity to theamino acid sequence of residues 181 to 419 of SEQ ID NO: 2, residues 1to 495 of SEQ ID NO: 4. Preferably, a full length GBS toxin receptorprotein comprises the amino acid sequence of residues 1 to 495 of SEQ IDNO: 2, residues 1 to 495 of SEQ ID NO: 4, or residues 1 to 536 of SEQ IDNO: 8, or an allelic variant thereof. The polypeptides of the inventioncan include amino acids in addition to the GBS toxin receptor protein,polypeptide, or polypeptide fragment. Such polypeptides typicallycomprise a heterologous polypeptide joined to a second polypeptidederived, as described above, from a GBS toxin receptor. Preferably theadditional amino acids are covalently linked to the amino-terminal orcarboxy-terminal terminus of the GBS toxin receptor protein,polypeptide, or polypeptide fragment.

Fragments or analogs of GBS toxin receptor can be prepared by those ofordinary skill in the art.

Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. For example, such functional domainsinclude domains conferring the property of induction of an inflammatoryresponse upon binding of GBS toxin to the GBS toxin receptor. GBS toxinmediates the binding and opsonization by C3 of endothelial cells thatexpress the GBS toxin receptor. Such domains can comprise the bindingsite for GBS toxin, in whole or in part, or domains otherwise essentialfor GBS toxin receptor structure and/or function. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known (Bowie etal. (1991) Science 253: 164). Computerized prediction methods, such as,for example, a hydropathy profile as provided by the “Soap” program inPC/GENE can be employed to identify putative structural and functionaldomains. Using the method of Klein, Kanehisa and DeLise, Biochim BiophysActa (1985) 815:468-476, the inventors have classified a sheep GBS toxinreceptor, SP55, as an integral protein with seven transmembrane segmentspredicted. Such a protein is also known colloquially in the art as a“7-spanner”. The predicted segments are set forth below in Table 3.

TABLE 3 Predicted Transmembrane Domains of SP55 Inner Outer Bound-Bound- aries aries Segment No. From To From To Sequence P:I odds* 1 232248 226 252 FFGIVGIIWFILWICLV 2.589323E−05 (232-248 of SEQ ID No. 4) 2369 385 365 389 LIGMIGPAIFLVAAGFI 1.007311E−03 (369-385 of SEQ ID No. 4)3 458 474 456 479 TVFCIAAAINVFGAIFF 2.482542E−03 (458-474 of SEQ ID No.4) 4 137 153 135 157 LLLGFGIFATAIFTLFT 7.564906E−03 (137-153 of SEQ IDNo. 4) 5  42  58  42  58 LAFLSFFGFFVLYSLRV 8.236557E−02 (42-58 of SEQ IDNo. 4) 6 328 344 328 345 GFLSAVPYLGCWLCMIL  .1925022 (328-344 of SEQ IDNo. 4) 7 390 406 390 407 SLAVAFLTISTTLGGFC  .8064944 (390-406 of SEQ IDNo. 4) *Relates hydrophobicity of integral sequence to thehydrophobicity of the peripheral sequence. An integral sequence with ahigher hydrophobicity number is more likely to be part of atransmembrane domain.

A computerized alignment of the amino acid sequences of GBS toxinreceptor in various organisms provides further guidance in preparingpreferred fragments. See, for example, Table 4 which compares the aminoacid sequence of residues 42 to 536 of a human GBS toxin receptor (HP59)(residues 42 to 536 of SEQ ID NO: 8) and a sheep GBS toxin receptor(SP55).

TABLE 4 Alignment of Human and Sheep GBS Toxin Receptor Amino AcidSequences

HPS5 - SEQ ID NO: 2 SP55 - SEQ ID NO: 4Thus, the foregoing examples demonstrate that those of skill in the artcan recognize sequence motifs and structural conformations that may beused to define structural and functional domains in a GBS toxin receptorsequence.

Although one class of preferred embodiments are fragments having amino-and/or carboxy-termini corresponding to amino acid positions nearfunctional domains borders, alternative fragments may be prepared. Thechoice of the amino- and carboxy-termini of such fragments rests withthe discretion of the practitioner and will be made based onexperimental considerations, such as ease of construction, stability toproteolysis, thermal stability, immunological reactivity, amino- orcarboxyl-terminal residue modification, or other considerations.Polypeptide fragments usually contain at least nine amino acids and cancontain any number of amino acids provided that the peptide fragment isat least about 80% identical to the corresponding fragment of SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:8. The human GBS toxin receptor has 41additional amino acids on the N-terminus compared to the sheep GBS toxinreceptor (compare SEQ ID NO:4 and SEQ ID NO:8). Analogs can compriseadditions or deletions of some or all of those 41 N-terminal aminoacids. N-terminal and C-terminal additions useful, e.g., forpurification and/or antibody recognition are also contemplated. Examplesinclude histidine tags, a FLAG (phenylalanine, leucine, alanine,guanine) epitope, fusion partners such as glutathione S transferase,chloramphenicol acetyltransferase (CAT), luciferase, β-galactosidase,and the like. Deletions of unconserved amino acids are alsocontemplated, provided that the structural integrity and/or bindingproperties of the GBS toxin receptor are not substantially compromised.

Analogs can also comprise amino acid substitutions, preferablyconservative substitutions. Also preferred are conservative and/ornon-conservative substitutions in regions having less shared identityamong various species. For example, a variant of a GBS toxin receptorcan comprise conservative and/or non-conservative substitutions of aminoacids corresponding to residues 2, 6, 10, 11, 16, 17, 24, 31, 44, 46,52, 53, 55, 74, 75, 81, 82, 84, 93, 102, 132, 133, 137, 144, 145, 148,149, 155, 159, 163, 165, 166, 179, 212, 219, 235, 236, 239, 242, 246,253, 254, 257, 259, 260, 271, 283, 285, 319, 324, 332, 333, 338, 355,362, 366, 377, 439, 442, 445, 450, 453, 461, 487, 488, 491 and 495 ofSEQ ID NO:4. Preferably the substitution is an amino acid present in thecorresponding position of SEQ ID NO:4 or SEQ ID NO:8. For example,referring to the alignment plot in Table 4, the amino acid correspondingto position 152 of SEQ ID NO:4 can be arginine (R), glutamine (Q), or aconservative or non-conservative substitution of R or Q, and preferablyis R or Q. Such regions can be identified by amino acid sequencealignment plots, such as that shown in Table 4. Preferred amino acidsubstitutions are those which: (1) reduce susceptibility to proteolysis,(2) reduce susceptibility to oxidation, (3) alter binding affinity forGBS toxin, and (4) confer or modify other physicochemical or functionalproperties of such analogs. Analogs can include various mutations of asequence other than the naturally-occurring peptide sequence, such as,for example, single or multiple amino acid substitutions.

A conservative amino acid substitution should generally notsubstantially change the structural characteristics of the parentsequence (e.g., a replacement amino acid should not tend to break ahelix that occurs in the parent sequence, disrupt disulfide bonds ordisrupt other types of secondary structure that characterizes the parentsequence). Examples of art-recognized polypeptide secondary and tertiarystructures are described in Proteins, Structures and MolecularPrinciples, (1984) Creighton (ed.), W.H. Freeman and Company, New York;Introduction to Protein Structure, (1991), C. Branden and J. Tooze,Garland Publishing, New York, N.Y.; and Thornton et al. (1991) Nature354: 105 (which are incorporated herein by reference). A conservativesubstitution is a “replacement of an amino acid in a polypeptide by onewith similar characteristics.” (McGraw-Hill Dictionary of Scientific andTechnical Terms, Fifth Edition, 1994, Sybil P. Parker, Editor in Chief).The structure and characteristics of naturally occurring amino acids haslong been known in the art (Biochemistry, Second Edition, Albert L.Lehninger, 1975, pages 71-76) For example, amino acids which are similarby virtue of their hydrophobic R groups are alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan, and methionine. Alanine,valine, leucine, and isoleucine are similar by virtue of their aliphaticR groups. Phenylalanine and tryptophan are similar by virtue of theiraromatic R groups. Glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine are similar by virtue of their uncharged polarR groups. Glycine and alanine are similar by virtue of their small size.Serine and threonine are similar by virtue of a hydroxyl in their Rgroup. Asparagine and glutamine differ by only one methyl group.Similarly, aspartic acid and glutamic acid differ by only one methylgroup, and they are similar by virtue of their acidic R groups. Lysine,arginine, and histidine are similar by virtue of their basic R groups.In addition, lysine and arginine are similar by virtue of the aminogroups on the end of the aliphatic chain in their R groups. Tyrosine andphenylalanine are similar by virtue of their aromatic groups. Aminosubstitutions commonly made in the art include a substitution of valinefor leucine or isoleucine, alanine for glycine, serine for threonine,asparagine for glutamine, aspartic acid for glutamic acid, and lysinefor arginine, tyrosine for phenylalanine, and vice versa.

Typically, one skilled in the art would generally refrain from changingamino acids that are conserved among the various GBS toxin receptors,but a conservative substitution might reasonably be made. For example,Table 4 guides one skilled in the art to avoid substitutions,particularly nonconservative substitutions, for amino acidscorresponding to residues 1, 3-5, 7-9, 12-15, 18-23, 26-30, 32-43, 45,47-51, 54, 56-73, 76-80, 83, 85-92, 94-101, 103-131, 134-136, 138-143,146-147, 150-154, 156-158, 160-162, 164, 167-178, 180-211, 213-218,220-234, 237-238, 240-241, 243-245, 247-252, 255-256, 258, 261-270,272-282, 284, 286-318, 320-323, 325-331, 334-337, 339-354, 356-361,363-365, 367-376, 378-438, 440-441, 443-444, 446-449, 451-452, 454-460,462-486, 489-490 and 492-494 of SEQ ID NO:4, which are conserved amongthe GBS toxin receptors shown in Table 4.

Tables 5 and 6 describe sequences within HP59 and SP55, respectively,that match predicted amidation, N-glycosylation, cAMP-phosphorylation,CK2-phosphosylation, myristylation (addition of unsaturated fatty acidmolecules), and PKC-phosphosylation sites (Omega 1.1 sequence analysisprogram). The information contained in these tables provides guidance toone skilled in the art for designing GBS toxin receptor variants andfragments. When designing polypeptide variants, for example, one maydecide to avoid substitutions in some or all of these regions. Whendesigning polypeptide fragments other than immunogenic polypeptidefragments, for example, one may opt to include some or all of theseregions.

TABLE 5 Putative Recognition Sites in HP59 Seq. ID NO: 8 Site Residues:Sequence AMIDATION 23-26 SGRR AMIDATION 138-141 TGKK ASN_GLYCOSYLATION100-103 NLSV ASN_GLYCOSYLATION 112-115 NTTL ASN_GLYCOSYLATION 118-121NRTS ASN_GLYCOSYLATION 136-139 NQTG ASN_GLYCOSYLATION 266-269 NWTYASN_GLYCOSYLATION 343-346 NWTF ASN_GLYCOSYLATION 398-401 NFSTCAMP_PHOSPHO_SITE 297-300 KRIS CK2_PHOSPHO_SITE 113-116 TTLECK2_PHOSPHO_SITE 114-117 TLED CK2_PHOSPHO_SITE 300-303 SHYECK2_PHOSPHO_SITE 493-496 TVGE MYRISTYL 66-71 GAPRAE MYRISTYL 167-172GGYVAS MYRISTYL 183-188 GILGTA MYRISTYL 213-218 GLGEGV MYRISTYL 246-251GAQLGT MYRISTYL 250-255 GTVISL MYRISTYL 378-383 GSWLCM MYRISTYL 427-432GCDYSL MYRISTYL 444-449 GGFCSS MYRISTYL 464-469 GILLGI MYRISTYL 468-473GITNTF PKC_PHOSPHO_SITE 23-25 SGR PKC_PHOSPHO_SITE 58-60 TDRPKC_PHOSPHO_SITE 78-80 SAR PKC_PHOSPHO_SITE 120-122 TSK PKC_PHOSPHO_SITE138-140 TGK PKC_PHOSPHO_SITE 310-312 SLR PKC_PHOSPHO_SITE 317-320 SQK

TABLE 6 Putative Recognition Sites in SP55 Seq. ID NO: 4 Site Residues:Sequence AMIDATION  97-100 TGKK ASN_GLYCOSYLATION 59-62 NLSVASN_GLYCOSYLATION 71-74 NTTA ASN_GLYCOSYLATION 77-80 NRTSASN_GLYCOSYLATION 95-98 NQTG ASN_GLYCOSYLATION 225-228 NWTYASN_GLYCOSYLATION 302-305 NWTF ASN_GLYCOSYLATION 357-360 NFSTCK2_PHOSPHO_SITE 11-14 SDGE CK2_PHOSPHO_SITE 73-76 TAKD CK2_PHOSPHO_SITE79-82 TSYE CK2_PHOSPHO_SITE 259-262 TPYE CK2_PHOSPHO_SITE 452-455 TIGEMYRISTYL 126-131 GGYVAS MYRISTYL 142-147 GIFATA MYRISTYL 162-167 GALVALMYRISTYL 172-177 GLGEGV MYRISTYL 205-210 GAQLGT MYRISTYL 209-214 GTVVSLMYRISTYL 337-342 GCWLCM MYRISTYL 386-391 GCDYSL MYRISTYL 403-408 GGFCSSMYRISTYL 423-428 GILLGI MYRISTYL 427-432 GITNTF PKC_PHOSPHO_SITE 17-19SDR PKC_PHOSPHO_SITE 37-39 SAR PKC_PHOSPHO_SITE 55-57 SLRPKC_PHOSPHO_SITE 73-75 TAK PKC_PHOSPHO_SITE 97-99 TGK PKC_PHOSPHO_SITE254-256 THK PKC_PHOSPHO_SITE 269-271 SLK PKC_PHOSPHO_SITE 276-278 SQK

In light of the foregoing, preferred polypeptides comprise an amino acidsequence of the formula:

AA1-AAn-AAmwherein:

AA1 is absent or is M;

AAn is a contiguous chain of 0 to 100 amino acids, preferably of 0 or 41amino acids, even more preferably of residues 2-42 of SEQ ID NO:8; and

AAm is a contiguous chain of 494 amino acids comprising AA43 throughAA536, wherein:

-   -   (1) each of AA43, AA47, AA51, AA52, AA57, AA58, AA65, AA66,        AA72, AA85, AA87, AA93, AA94, AA96, AA115, AA116, AA122, AA123,        AA125, AA134, AA143, AA173, AA174, AA178, AA185, AA186, AA189,        AA190, AA196, AA200, AA204, AA206, AA207, AA220, AA253, AA260,        AA276, AA277, AA280, AA283, AA287, AA294, AA295, AA298, AA300,        AA301, AA312, AA324, AA326, AA360, AA365, AA373, AA374, AA379,        AA396, AA403, AA407, AA418, AA480, AA483, AA486, AA491, AA494,        AA502, AA528, AA529, AA532 and AA536 is an essential amino acid        or a modified amino acid and preferably is an amino acid residue        corresponding to:        -   (a) residue 43, 47, 51, 52, 57, 58, 65, 66, 72, 85, 87, 93,            94, 96, 115, 116, 122, 123, 125, 134, 143, 173, 174, 178,            185, 186, 189, 190, 196, 200, 204, 206, 207, 220, 253, 260,            276, 277, 280, 283, 287, 294, 295, 298, 300, 301, 312, 324,            326, 360, 365, 373, 374, 379, 396, 403, 407, 418, 480, 483,            486, 491, 494, 502, 528, 529, 532 and 536, respectively, of            SEQ ID NO:8;        -   (b) residue 2, 6, 10, 11, 16, 17, 24, 25, 31, 44, 46, 52,            53, 55, 74, 75, 81, 82, 84, 93, 102, 132, 133, 137, 144,            145, 148, 149, 155, 159, 163, 165, 166, 179, 212, 219, 235,            236, 239, 242, 246, 253, 254, 257, 259, 260, 271, 283, 285,            319, 324, 332, 333, 338, 355, 362, 366, 377, 439, 442, 445,            450, 453, 461, 487, 488, 491 and 495, respectively of SEQ ID            NO:4; or        -   (c) a conservative substitution thereof;    -   (2) each of AA44-AA46, AA48-AA50, AA53-AA56, AA59-AA64,        AA67-AA71, AA73-AA84, AA86, AA88-AA92, AA95, AA97-AA114,        AA117-AA121, AA124, AA126-AA133, AA135-AA142, AA144-AA172,        AA175-AA177, AA179-AA184, AA187-AA188, AA191-AA195, AA197-AA199,        AA201-AA203, AA205, AA208-AA219, AA221-AA252, AA254-AA259,        AA261-AA275, AA278-AA279, AA281-AA282, AA284-AA286, AA288-AA293,        AA296-AA297, AA299, AA302-AA311, AA313-AA323, AA325,        AA327-AA359, AA361-AA364, AA366-AA372, AA375-AA378, AA380-AA395,        AA397-AA402, AA404-AA406, AA408-AA417, AA419-AA478, AA481-AA482,        AA484-AA485, AA487-AA490, AA492-AA493, AA495-AA501, AA503-AA527,        AA530-AA531 and AA533-AA535 is        -   (a) residue 44-46, 48-50, 53-56, 59-64, 67-71, 73-84, 86,            88-92, 95, 97-114, 117-121, 124, 126-133, 135-142, 144-172,            175-177, 179-184, 187-188, 191-195, 197-199, 201-203, 205,            208-219, 221-252, 254-259, 261-275, 278-279, 281-282,            284-286, 288-293, 296-297, 299, 302-311, 313-323, 325,            327-359, 361-364, 366-372, 375-378, 380-395, 397-402,            404-406, 408-417, 419-478, 481-482, 484-485, 487-490,            492-493, 495-501, 503-527, 530-531 and 533-535,            respectively, of SEQ ID NO:8; or        -   (b) a conservative substitutions thereof; and    -   (3) AA315 through AA367 are optionally absent.

Preferred polypeptides comprise the amino acid sequence of SEQ ID NO:4,SEQ ID NO:8 or an amino acid sequence which varies from that sequenceonly at the specific residues which are not conserved between the sheepGBS toxin receptor (SEQ ID NO:4) and the human GBS toxin receptor (SEQID NO:8). Of those variations, the most preferred variations are thoseresulting in a polypeptide encoded by SEQ ID NO:11. Even more preferredvariations are those amino acids in the corresponding positions of theamino acid sequence of SEQ ID NO:4. Particularly preferred arepolypeptides comprising an amino acid sequence that differs from SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:8 at no more than about 20% of the aminoacid residues, with increasing preference for no more than about 10%,5%, 1%, with one to zero amino acid differences being most preferred.

Besides targeting specific amino acids for change, analogs of GBS toxinreceptor can also be prepared by techniques involving activityselection, such as, for example, phage display, directed evolution, DNAshuffling, and homologous in procaryotes or eucaryotes of genes fromdifferent species, as described in part in U.S. Pat. Nos. 5,605,793;5,830,721; 5,811,238; 5,837,458; 5,093,257; 5,223,409; 5,403,484;5,571,698; and 5,837,500, which are incorporated herein by reference.

Any variant or fragment of the human and sheep GBS toxin receptorsdescribed herein can be tested for the requisite activity by determiningwhether the variant or fragment can bind GBS toxin.

These polypeptides provide reagents useful in drug discovery andpurification and can be used in various in vitro assays, preferably whenexpressed on the surface of a cell, e.g., a stable transfected cell. Forexample, assays such as binding assays can be used to screen testcompounds, including polysaccharides and other compounds, for theirability to bind the GBS toxin receptor. Assays can identify potentialdrug candidates that block GBS toxin binding to the GBS toxin receptor.Such drugs are useful for preventing and/or treating early onset diseasein neonatal humans. Some polypeptides can be used to competitivelyinhibit binding GBS toxin to a GBS toxin receptor.

The polypeptides of the invention can be used to affinity purify GBStoxin, a GBS toxin chimeric compound, and other polysaccharides orcompounds which can bind the GBS toxin receptor.

The polypeptides can also be used to develop a method of targeting acytotoxic agent for delivery to a cell that expresses a GBS toxinreceptor. For example, a cytotoxic agent can be coupled to a moleculethat binds a GBS toxin receptor for selective delivery to theneovasculature of a growing tumor. Such a delivery system would permit ahighly concentrated, localized attack on a growing tumor, whileminimizing the adverse systemic side effects encountered with mostchemotherapeutics. In one instance, the cytotoxic agent can be GBStoxin, which, upon binding to GBS toxin receptor, induces aninflammatory response as described in Hellerqvist et al., Angiogenesis:Molecular Biology. Clinical Aspect, Edited by M. E. Maragoudakis et al.,Plenum Press, New York 1994, pp. 265-269. In a similar manner, selectivedelivery of a therapeutic agent to a cell that expresses a GBS toxinreceptor could be used advantageously to treat tumors, rheumatoidarthritis or neural injury, or to facilitate wound healing.

The polypeptides of the invention can also be used to screen for and/ordesign a GBS toxin mimetic with improved therapeutic properties, suchas, for example, improved ability to inhibit hypoxia-inducedneovascularization or angiogenesis. Such mimetics are useful in thetreatment and prevention of conditions resulting from hypoxia-inducedneovascularization or angiogenesis, such as, for example, tumor growth,scarring during wound healing, gliosis during repair of neural injury,reperfusion injury, restenosis, rheumatoid arthritis, psoriasis, otherchronic inflammatory diseases characterized by angiogenesis, etc.Therapeutic properties can be improved by enhancing biologicalstability, affinity for the GBS toxin receptor, complement bindingactivity, reducing antigenicity, etc.

The polypeptides of the invention can also be used to generateantibodies for various therapeutic and research purposes. Thepolypeptides of the invention can be used to immunize rabbits, mice,goats, chickens, or other animals known in the art to be amenable tosuch immunization. Monoclonal antibodies are generally preferred butpolyclonal antibodies can also be used, provided that detection ofbinding of the GBS toxin receptor antibody to the GBS toxin receptor ispossible. The production of non-human monoclonal antibodies, e.g.,murine, is well known (see, e.g., Harlow et al., Antibodies A LaboratoryManual, Cold Spring Harbor Press, pp. 139-240, 1989, incorporated hereinby reference). As it may be difficult to generate human monoclonalantibodies to a human receptor or binding domain polypeptide, it may bedesirable to transfer antigen binding regions of non-human monoclonalantibodies, e.g. the F(ab′)₂ or hypervariable regions or murinemonoclonal antibodies, to human constant regions (Fc) or frameworkregions by recombinant DNA techniques to produce substantially humanmolecules. Such methods are generally known and are described in, e.g.,U.S. Pat. Nos. 4,816,397 and 4,946,778, and EP publications 173,494 and239,400. Alternatively, one may isolate DNA sequences which code for ahuman monoclonal antibody or portions thereof that specifically bind tothe receptor protein by screening a DNA library from human B cellsaccording to the general protocol outlined in WO 90/14430, and thencloning and amplifying the sequences which encode the antibody (orbinding fragment) of the desired specificity.

Usually, polypeptides used for producing antibodies are the full-lengthreceptor or receptor fragments designed from putative extracellulardomains identified by a variety of methods known in the art, includingcomputer programs which predict secondary and tertiary structure of apolypeptide based upon its primary amino acid sequence. Another methodfor designing antigenic peptides utilizes computer programs that predictthe high points of hydrophilicity within a particular primary amino acidsequence. For example, using the method of Happ and Woods, Proc. Natl.Acad. Sci. USA (1981) 78:3824-3829, via the “Antigen” program inPC/GENE, the inventors identified 3 regions of high hydrophilicity,shown below in Table 7, and used the results to design antigenicpeptides to be used in the preparation of antibodies against GBS toxinreceptor (see Example 4).

TABLE 7 High Points of Hydrophilicity in SP55 No. Ah Sequence 1 2.05Glu-Glu-Gly-Ser-Asp-Arg (14-19 of SEQ ID No. 2) 2 1.52Lys-Asp-Asn-Arg-Thr-Ser (75-80 of SEQ ID No. 2) 3 1.33Arg-Ala-Pro-Arg-Ala-Glu (25-30 of SEQ ID No. 2) Ah = Averagehydrophilicity.

Antibodies that recognize various portions of the intact GBS toxinreceptor can be used to further investigate structure and function ofthe receptor. The polypeptides of the invention can give rise toantibodies that recognize a variety of forms of GBS toxin receptor,including, but not limited to, intact GBS toxin receptor expressed on acell surface, denatured GBS toxin receptor or non-denatured GBS toxinreceptor, and GBS toxin receptor purified away from cellular componentsor GBS toxin receptor contained in a cell lysate. GBS toxin receptorantibodies can be used to study species differences as well as GBS toxinreceptor expression levels in various cell types.

Antibodies that recognize a portion or all of an extracellular domainare particularly useful as a diagnostic for the monitoring of tumorgrowth and metastasis, for the detection or identification of a chronicinflammatory condition, such as, for example, rheumatoid arthritis orpsoriasis, and for the detection of other medical conditions arising dueto hypoxia-driven angiogenesis, such as, for example, restenosis.Typically, such antibodies can be employed in a variety of standardresearch and diagnostic techniques, including, but not limited to,western blot, immunoprecipitation, ELISA, radioimmunoassay (RIA),BIACOR®, enzyme-linked-immunoassay (EIA), immunofluorescence,fluorescence activated cell sorting (FACS), and in vivo diagnosticimaging systems such as magnetic resonance imaging (MRI), nuclearmagnetic resonance (NMR), computerized axial tomography (CAT) scan, andposition emission tomography (PET), etc.

In addition, antibodies that block the binding of GBS toxin to a GBStoxin receptor can be used for the treatment or prevention of earlyonset disease in a neonatal human. Such antibodies can directly orindirectly block the GBS toxin binding site on the GBS toxin receptor.

In one embodiment, the GBS toxin receptor protein is naturally occurringand can be isolated from a cell extract by protein purificationtechniques known in the art, such as, for example, ion exchange columnchromatography, high performance liquid chromatography. (HPLC), reversedphase HPLC, or affinity chromatography using antibodies that recognizethe GBS toxin receptor.

Alternatively, the isolated proteins and polypeptides are expressedusing polynucleotides encoding the polypeptide(s) of the invention inoperative association with an appropriate control sequence including apromoter in an expression vector suitable for expression, preferably ina mammalian cell, and also in bacterial, insect, or yeast cells.

Usually, the GBS toxin receptor polynucleotide or a fragment thereof canbe expressed in a mammalian system. Such expression will usually dependon a mammalian promoter, which is any DNA sequence capable of bindingmammalian RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g. structural gene) into mRNA.Usually, a promoter will have a transcription initiation region which isusually placed proximal to the 5′ end of the coding sequence. Thistranscription initiation region typically includes an RNA polymerasebinding site and a transcription initiation site.

Vectors suitable for replication in mammalian cells are known in theart, and can include viral replicons, or sequences that ensureintegration of the sequence encoding PAK65 into the host genome.Suitable vectors can include, for example, those derived from simianvirus SV40, retroviruses, bovine papilloma virus, vaccinia virus, andadenovirus.

A suitable vector, for example, is one derived from vaccinia viruses. Inthis case, the heterologous DNA is inserted into the vaccinia genome.Techniques for the insertion of foreign DNA into the vaccinia virusgenome are known in the art, and utilize, for example, homologousrecombination. The insertion of the heterologous DNA is generally into agene which is non-essential in nature, for example, the thymidine kinasegene (tk), which also provides a selectable marker. Plasmid shuttlevectors that greatly facilitate the construction of recombinant viruseshave been described (see, for example, Mackett et al. (1984);Chakrabarti et al. (1985); Moss (1987)). Expression of the heterologouspolypeptide then occurs in cells or individuals which are immunized withthe live recombinant vaccinia virus.

Such suitable mammalian expression vectors usually contain one or moreeukaryotic transcription units that are capable of facilitatingexpression in mammalian cells. The transcription unit is comprised of atleast a promoter element to mediate transcription of foreign DNAsequences. Suitable promoters for mammalian cells are known in the artand include viral promoters such as those from simian virus 40 (SV40)(Subramani et al., Mol Cell. Biol. 1:854-864, 1981), cytomegalovirus(CMV) (Boshart et al., Cell 41:521-530, 1985), Rous sarcoma virus (RSV),adenovirus (ADV) (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-1319,1982), and bovine papilloma virus (BPV), as well as cellular promoters,such as a mouse metallothionein-1 promoter (U.S. Pat. No. 4,579,821), amouse VK promoter (Bergman et al., Proc. Natl. Acad. Sci. USA81:7041-7045, 1993; Grant et al., Nuc. Acids Res. 15:5496, 1987), and amouse VH promoter (Loh et al., Cell 33:85-93, 1983).

The optional presence of an enhancer element (enhancer), combined withthe promoter elements described herein, will typically increaseexpression levels. An enhancer is any regulatory DNA sequence that canstimulate transcription up to 1000-fold when linked to endogenous orheterologous promoters, with synthesis beginning at the normal mRNAstart site. Enhancers are also active when they are placed upstream ordownstream from the transcription initiation site, in either normal orflipped orientation, or at a distance of more than 1000 nucleotides fromthe promoter (Maniatis et al. (1987) Science 236:1237; Alberts et al.(1989) Molecular Biology of the Cell, 2nd ed.). Enhancer elementsderived from viruses can be particularly useful, because they typicallyhave a broader host range. Examples useful in mammalian cells includethe SV40 early gene enhancer (Dijkema et al (1985) EMBO J. 4:761) andthe enhancer/promoters derived from the long terminal repeat (LTR) ofthe Rous Sarcoma Virus (Gorman et al. (1982b) Proc. Natl. Acad. Sci.79:6777), from human cytomegalovirus (Boshart et al. (1985) Cell 41:521)as well as the mouse μ enhancer (Gillies, Cell 33:717-728, 1983).Additionally, some enhancers are regulatable and become active only inthe presence of an inducer, such as a hormone or metal ion(Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al.(1987) Science 236:1237).

In addition, the transcription unit can also be comprised of atermination sequence and a polyadenylation signal which are operablylinked to the GBS toxin receptor coding sequence. Polyadenylationsignals include, but are not limited to, the early or latepolyadenylation signals from SV40 (Kaufman and Sharp), thepolyadenylation signal from the Adenovirus 5 E1B region and the humangrowth hormone gene terminator (DeNoto et al., Nuc. Acids Res.9:3719-3730, 1981).

Sequences that cause amplification of the gene may also be desirable, asare sequences which encode selectable markers. Selectable markers formammalian cells are known in the art, and include, for example,thymidine kinase, dihydrofolate reductase (together with methotrexate asa DHFR amplifier), aminoglycoside phosphotransferase, hygromycin Bphosphotransferase, asparagine synthetase, adenosine deaminase, andantibiotic resistant genes such as neomycin.

A GBS toxin receptor, or fragment thereof, can be expressed on thesurface of a cell, or can be expressed in soluble or secreted form.Expression on the surface of the cell can be achieved, for example, byincluding a secretory leader operably linked to a nucleic acid sequenceencoding the desired receptor fragment and at least one transmembranedomain. The secretory leader can be that encoded by the GBS toxinreceptor gene, or can be a heterologous leader sequence commonly used inthe art, such as, for example, the leader sequence ofSchizosaccharomyces pombe pho1+acid phosphatase (Braspenning et al.,Biochem Biophys Res. Commun (1998) 245:166-71), the leader sequence ofhuman interleukin-2 (IL-2) gene (Sasada et al., Cell Struct Funct (1988)13:129-141). Expression in soluble or secreted form can be achieved, forexample, by excluding from the gene construct nucleic acid sequencesencoding a transmembrane domain. In some instances, solubility and/orsecretion are achieved by the use of a fusion partner, such as, forexample, chloramphenicol acetyltransferase (CAT), β-galactosidase, andother genes readily expressed in the selected host cell.

The vector that encodes GBS toxin receptor can be used fortransformation of a suitable mammalian host cell. Transformation can beby any known method for introducing polynucleotides into a host cell,including, for example packaging the polynucleotide in a virus andtransducing a host cell with the virus or by transfection proceduresknown in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (these patents are incorporated herein byreference). The transformation procedure used depends upon the host tobe transformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), N1E-115 (Liles et al., J. Biol. Chem.261:5307-5313, 1986), PC 12 human hepatocellular carcinoma cells (e.g.,Hep G2), and a number of other cell lines, such as insect derived celllines IF9 and IF21. Cell lines of particular preference are thoseexpressing recombinant GBS toxin receptor constructs constitutively,lines which subsequently develop characteristics of a transformed cell,and lines which more preferably express GBS toxin receptor or fragmentson the cell surface. Particularly preferred are ECV cells (a bladdercarcinoma cell line originally referred to in the scientific literatureas an endothelial cell line), human umbilical vein endothelial cells(HUVEC), bovine, sheep, and human adrenal medulla endothelial cells.

Recombinant GBS toxin receptor or fragments thereof can be produced byculturing host cells expressing the receptor or fragment in a suitableculture medium and under appropriate cell culture conditions. Culturemedia and conditions are variable depending on the requirements of aparticular host cell line and are well known in the art. Typically,cells are cultured at 37° C. in a cell culture incubator with a fixedamount of C02, usually in the range of 5-10%.

In another embodiment, the polypeptide fragments can be synthesizedchemically by techniques well known in the art, such as solid-phasepeptide synthesis (Stewart et al., Solid Phase Peptide Synthesis, W.H.Freeman Co., San Francisco (1963)); Merrifield, J Am Chem Soc85:2149-2154 (1963)). These and other methods of peptide synthesis arealso exemplified by U.S. Pat. Nos. 3,862,925, 3,842,067, 3,972,859, and4,105,602. The synthesis can use manual synthesis techniques orautomatically employ, for example, an Applied BioSystems 430A or 431APeptide Synthesizer (Foster City, Calif.) following the instructionsprovided in the instruction manual supplied by the manufacturer. It willbe readily appreciated by those having ordinary skill in the art ofpeptide synthesis that the intermediates which are constructed duringthe course of synthesizing the present analog compounds are themselvesnovel and useful compounds and are thus within the scope of theinvention.

In addition to polypeptides consisting only of naturally-occurring aminoacids, peptidomimetics are also provided. Peptide analogs are commonlyused in the pharmaceutical industry as non-peptide drugs with propertiesanalogous to those of the template peptide. These types of non-peptidecompounds are termed “peptide mimetics” or “peptidomimetics” (Fauchere,J. (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem 30: 1229, which areincorporated herein by reference) and are usually developed with the aidof computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity) but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH2NH—, —CH2S—,—CH2-CH2-, —CH═CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and —CH2SO—,by methods known in the art and further described in the followingreferences: Spatola, A. F. in “Chemistry and Biochemistry of AminoAcids, Peptides, and Proteins,” B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983); Vol. 1, Issue 3, “Peptide Backbone Modifications”(general review); Morley, J. S., Trends Pharm Sci (1980) pp. 463-468(general review); Hudson, D. et al., Int J Pept Prot Res (1979)14:177-185 (—CH2NH—, CH2CH2-); Spatola, A. F. et al., Life Sci (1986)38:1243-1249 (—CH2-S); Hann, M. M., J Chem Soc Perkin Trans I (1982)307-314 (—CH—CH—, cis and trans); Almquist, R. G. et al., J. Med Chem(1980) 23:1392-1398 (—COCH2-); Jennings-White, C. et al., TetrahedronLett (1982) 23:2533 (—COCH2-); Szelke, M. et al., European Appin. EP45665 (1982) CA: 97:39405 (1982) (—CH(OH)CH2-); Holladay, M. W. et al.,Tetrahedron Lett (1983) 24:4401-4404 (—C(OH)CH2-); and Hruby, V. J.,Life Sci (1982) 31:189-199 (—CH2-S—); each of which is incorporatedherein by reference. A particularly preferred non-peptide linkage is—CH2NH—. Such peptide mimetics may have significant advantages overpolypeptide embodiments, including, for example: more economicalproduction, greater chemical stability, enhanced pharmacologicalproperties (half-life, absorption, potency, efficacy, etc.), alteredspecificity (e.g., a broad-spectrum of biological activities), reducedantigenicity, and others. Labeling of peptidomimetics usually involvescovalent attachment of one or more labels, directly or through a spacer(e.g., an amide group), to non-interfering position(s) on thepeptidomimetic that are predicted by quantitative structure-activitydata and/or molecular modeling. Such non-interfering positions generallyare positions that do not form direct contacts with GBS toxin (e.g., arenot contact points in the GBS toxin binding domain of the GBS toxinreceptor). Derivitization (e.g., labelling) of peptidomimetics shouldnot substantially interfere with the desired biological orpharmacological activity of the peptidomimetic.

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387,incorporated herein by reference); for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

The invention also provides a complex comprising a GBS toxin bound to amammalian GBS toxin receptor or a fragment of a mammalian GBS toxinreceptor. Preferably, the complex comprises a GBS toxin bound to a GBStoxin receptor polypeptide described above that can bind GBS toxin.Typically, a complex is formed by contacting a GBS toxin with such apolypeptide under conditions that permit specific binding of the GBStoxin to the polypeptide. The GBS toxin can be labeled or unlabeled. Thepolypeptide can be present on the surface of a cell, or immobilized in awell or on a bead, or the polypeptide can be present in solution.

Detection Methods

Yet another aspect of the invention provides methods for detecting ormonitoring a variety of medical conditions characterized by pathologicand/or hypoxia-driven angiogenesis or neovascularization. Examplesinclude, but are not limited to, early onset disease in the neonate, andthe progression of cancers involving tumors.

Early onset disease can be diagnosed by detecting the presence orabsence of GBS toxin in a patient. One method of detection is acompetition assay that determines the effect of a suspected sample onthe formation of a complex between GBS toxin and a GBS toxin receptor orfragment thereof. For example, the method comprises contacting a controlGBS toxin with a GBS toxin receptor polypeptide, in the presence andabsence of a sample suspected of containing GBS toxin and underconditions that permit specific binding of the GBS toxin to thepolypeptide, and comparing the amount of complex formation achieved inthe presence of the suspected sample to the amount of complex formationachieved in the absence of the suspected sample. Preferably, the controlGBS toxin is substantially purified and of a known concentration.Preferably, the control GBS toxin further comprises a label. Suitablelabels include, but are not limited to, radioisotopes, chromophores,fluorophores, biotin, avidin, and other labels used by one skilled inthe art. Another method directly measures, rather than by competitionwith a control GBS toxin, complex formation between GBS toxin present ina suspected sample and a GBS toxin receptor polypeptide.

Pathologic vasculature can be detected in a mammalian tissue bydetecting the presence or absence of GBS toxin receptor in the region ofa tumor, with the presence of GBS toxin receptor being indicative of thepresence of pathologic vasculature. The method can be used to monitortumor growth or metastasis. One method of detection involves the use ofmolecules, e.g. antibodies, that specifically bind to a GBS toxinreceptor, preferably an extracellular domain of GBS toxin receptor.Typically, the method comprises administering, to a mammalian tissue,e.g. in a mammal having a cancerous tumor, e.g., an antibody thatrecognizes a GBS toxin receptor, and detecting specific binding of theantibody. Typically, the antibody is a labeled antibody. Preferably, theobservations are quantitative and can be visual.

During surgery, the margin of a tumor can be visualized by any of anumber of imaging techniques known in the art and described above. Theimaging of the tumor is effected by detecting the binding of a labeledantibody or other molecules to the GBS toxin receptor on the pathologicvasculature of a tumor. This type of surgery is also known as virtualsurgery because while performing the surgery, the surgeon views thetumor indirectly on an imaging screen.

Drug Discovery

A fourth aspect of the invention provides methods, using thepolypeptides of the invention, of identifying drug candidates for thetreatment of medical conditions characterized by hypoxia-drivenangiogenesis or neovascularization. Preferred compounds are competitiveinhibitors of GBS toxin binding to a GBS toxin receptor or inhibit GBStoxin receptor activity. Particularly preferred are compounds thatinhibit the first phosphorylation step in the signal transductionpathway. Compounds can be produced by a variety of random drug designmethods commonly known in the art, such as, for example, combinatorialchemistry (U.S. Pat. No. 5,646,285; U.S. Pat. No. 5,639,603), peptidelibraries (U.S. Pat. No. 5,591,646; U.S. Pat. No. 5,367,053; U.S. Pat.No. 5,747,334), phage display (U.S. Pat. No. 5,403,484; U.S. Pat. No.5,223,409), SELEX® (U.S. Pat. No. 5,773,598; U.S. Pat. No. 5,763,595;U.S. Pat. No. 5,763,566), and combinatorial carbohydrate chemistry(Hirschmann et al., J Med Chem (1996) 39:2441-2448; Hirschmann et al., JMed Chem (1998) 41:1382-1391; Sofia M J, Mol Divers (1998) 3:75-94; U.S.Pat. No. 5,780,603; U.S. Pat. No. 5,756,712)

An alternative approach is rational drug design with the intent ofproducing a GBS toxin mimetic or a GBS toxin receptor mimetic withimproved therapeutic properties using techniques such as x-raycrystallography, nuclear magnetic resonance (NMR) correlation spectra(U.S. Pat. No. 5,698,401), computer assisted molecular modeling (U.S.Pat. No. 5,579, 250; U.S. Pat. No. 5,612,895; U.S. Pat. No. 5,680,331,Cooper et al., J. Comput.-Aided Mol. Design, 3:253-259 (1989); Brent etal., J. Comput.-Aided Mol. Design 2:311-310 (1988)) and other methods ofrational drug design known in the art. FIG. 1 provides a broad overviewof some of the main steps in some of the rational drug design methods ofthe present invention. For example, one approach to rational drug designinvolves a computer program, such as INSIGHTII (available from BisoymTechnologies, 10065 Barnes Canyon Road, San Diego, Calif.) to identifyactive sites in proteins by homology-based modeling. This methodfacilitates the modeling of a protein by using a similar protein whosestructure is well known. Commercial software containing searchalgorithms for three dimensional database comparisons are available fromvendors such as Day Light Information Systems, Inc., Irvine, Calif.92714, and Molecular Design Limited, 2132 Faralton Drive, San Leandro,Calif. 94577.

In one embodiment, the compound can bind the GBS toxin receptor andinduce an inflammatory response in a manner similar to the binding ofGBS toxin to the GBS toxin receptor. Such compounds can be used, forexample, as a drug to target an inflammatory response to the developingvasculature of a tumor.

In another embodiment, the compound can bind the GBS toxin receptor withor without inducing an inflammatory response, preferably withoutinducing an inflammatory response. In one instance, the compound can beused as a vehicle to target pathological neovasculature for treatmentwith a cytotoxic agent. For example, the cytotoxic agent can bechemically coupled to the compound to form a chimeric drug. Suchchimeric drugs can be used in the treatment of tumors, rheumatoidarthritis, wound healing, spinal cord injury, and other conditionscharacterized by hypoxia-driven angiogenesis or neovascularization. Inanother instance, the compound can be used directly to competitivelyinhibit binding of GBS toxin to a GBS toxin receptor. Such compounds canbe used in the treatment of early-onset disease in the neonate.

In a third embodiment, the compound can bind GBS toxin and can be usedin the treatment of early-onset disease in the neonate.

The polynucleotides of the invention can be expressed in randommutagenesis systems such as phage display or the yeast two-hybrid systemfor the synthesis and identification of mutant peptide GBS toxinreceptor polypeptides that bind GBS toxin. Alternatively, immobilized orsoluble GBS toxin receptor fragments of the invention can be used toscreen combinatorial peptide and combinatorial chemical libraries andnon-random recombinant and synthetic peptides and other compounds (suchas non-peptide molecules) for GBS toxin receptor binding. Compounds thatbind GBS toxin or GBS toxin receptor can then be further characterizedin a functional assay for any of the activities described above in orderto identify a drug candidate for the treatment of medical conditionsinvolving angiogenesis or neovascularization.

A compound which inhibits binding of GBS toxin to a GBS toxin receptorcan be identified by combining a test compound with a mammalian GBStoxin receptor or fragment thereof capable of binding GBS toxin, underconditions that permit specific binding of GBS toxin to the GBS toxinreceptor or fragment, and determining the amount of inhibition by thecompound of the binding of GBS toxin to the GBS toxin receptor orfragment.

In a preferred embodiment, the GBS toxin receptor or fragment isexpressed by a cell, preferably on the cell surface. The cells arecontacted with labeled GBS toxin in the presence or absence of the testcompound. A change in the binding of GBS toxin to the GBS toxin receptoris then determined. Alternatively, the GBS toxin is unlabeled and anantibody that recognizes GBS toxin is labeled instead. The labeledantibody is used to measure inhibition by a compound of GBS toxinbinding to the GBS toxin receptor or fragment. In another embodiment,the GBS toxin receptor or fragment is not associated with a cell, but isinstead coupled to a matrix, such as, for example, a well in amicrotiter plate or a bead. Additional suitable solid supports includelatex, polystyrene beads (Interfacial Dynamics Corp. Portland, Oreg.),magnetic particles (Advanced Magnetics, Cambridge, Mass.) and nylonballs (Hendry et al., J. Immunological Meth., 35:285-296, 1980). Thereceptor or fragment can be coupled to the matrix directly or indirectlythrough an antibody, coupled to the matrix, that binds the receptorfragment. In a third embodiment, the GBS toxin receptor or fragment issoluble and can be immunoprecipitated with an antibody that recognizesthe receptor or fragment.

A preferred method for identifying a compound which binds a mammalianGBS toxin receptor comprises the steps of (1) combining a test compoundwith a GBS toxin receptor or fragment thereof under conditions thatallow specific binding to occur, and (2) detecting a complex formedbetween the test compound and the GBS toxin receptor or fragment. Apreferred method is a competition assay which determines the ability ofthe test compound to compete for binding to the GBS toxin receptor orfragment. In such an assay, GBS toxin is combined with the GBS toxinreceptor or fragment in the presence or absence of the test compound.Decreased specific binding of GBS toxin in the presence versus theabsence of the test compound is indicative of the ability of the testcompound to bind a mammalian GBS toxin receptor. Another methodcomprises combining a control compound with the GBS toxin receptor orfragment under the same conditions as the test compound and comparingthe amount of complex formation between the test compound or the controlcompound and the GBS toxin receptor or fragment thereof. Preferably, thetest compound and/or the control compound are labeled. The test compoundcan be any of a number of classes of compounds, such as for example,small organic molecules (such as those used for and obtained bycombinatorial chemistry), polysaccharides, polypeptides, RNA,antibodies, and single chain antibodies. In a preferred embodiment, thepolypeptide is expressed by a cell, preferably on the surface of thecell, and preferably by a stable transfected cell. Such a system isparticularly useful for testing the effectiveness of a chimeric compoundcomprising a cytotoxic agent. The cytotoxic activity of the compound canbe determined by exposing a cell expressing the GBS toxin receptor onthe cell surface to the test chimeric compound and detecting signs ofcytotoxicity. One could detect such signs by a viability stain of thecell, by detecting apoptosis (for example, by a DNA ladder assay or aTUNEL™ stain, which binds to broken DNA), by measuring tritiatedthymidine incorporation into the cell, and by quantitatingkinase-dependent phosphorylation (e.g., using phosphoantibodies orvarious phosphoimaging techniques).

In another embodiment, the invention provides a method for identifyingan inhibitor of GBS toxin receptor. The method comprises incubating testcells in the presence and absence of a test compound. The test cellsexpress GBS toxin receptor or a fragment thereof having GBS toxinreceptor activity (e.g., a fragment that increases the proliferation ormigration of the expressing cells relative to control cells of the samecell type that do not express the fragment). The test cells areincubated under conditions in which the cells incubated in the absenceof the test compound can proliferate or migrate. Control cells that donot express the GBS toxin receptor or fragment proliferate or migrateless than cells that express the GBS toxin receptor or fragment. Theproliferation or migration (also referred to herein as motility) of thetest cells incubated in the presence or absence of the test compound iscompared. Less proliferation or migration in the presence of the testcompound than in the absence of the test compound is indicative of thetest compound being an inhibitor of the GBS toxin receptor. Preferably,as a control to determine whether the test compound specificallyinhibits the GBS toxin receptor, the proliferation or migration ofcontrol cells in the presence and absence of the test compound is alsocompared. In the absence of a difference in the proliferation ormigration of control cells incubated in the presence or absence of thetest compound, decreased proliferation or migration in test cellsexposed to the test compound relative to test cells not exposed to thetest compound is indicative of specific inhibition of the GBS toxinreceptor. It will be readily apparent that the control portions of themethod need not be performed contemporaneously with the test portions ofthe method. For example, control cells can be incubated with a batteryof test compounds to determine cellular effects of the test compoundsprior to incubating the test cells with the test compounds. Motility ormigration can be determined by detecting movement of cells on a culturedish. Proliferation can be detected in a number of ways, including, butnot limited to, measuring tritiated thymidine incorporation, cellcounts, apoptosis assays, and viability assays. Preferred cells includecells transfected with GBS toxin receptor, preferably endothelial cellstransfected with GBS toxin receptor, even more preferably vascularendothelial cells or microvascular endothelial cells. Primary cells thatexpress GBS toxin receptor are also preferred, for example, endothelialcells that have been passaged in cell culture, at confluence, no morethan 8 or 9 times. A preferred class of test compounds includes kinaseinhibitors, preferably cAMP-dependent kinase inhibitors, PKC inhibitors,and CK2 inhibitors, which can be used as a starting point for developingmore specific GBS toxin receptor inhibitors. Another class of compoundsincludes antibodies specific for GBS toxin receptor. Particularlypreferred are single chain antibodies, preferably a collection of singlechain antibodies that recognize various epitopes on the GBS toxinreceptor. Less preferred are divalent antibodies specific for thebinding site of the GBS toxin receptor ligand because they may triggerthe signal transduction cascade upon dimerization.

Another embodiment of the invention is a method of identifying aninhibitor of endothelial cell proliferation or migration, which areessential components of angiogenesis. The method basically comprises thesteps described in the preceding paragraph and uses endothelial cells.

Yet another embodiment of the invention is a method of identifying atherapeutic compound for the treatment or prevention of a medicalcondition characterized by pathologic or hypoxia-driven angiogenesis orneovascularization. The method basically comprises the steps describedabove and uses cells from tissues derived from mammals afflicted withthe medical condition or cells that serve as a model for afflictedtissue.

A preferred method for designing a compound which inhibits binding of aGBS toxin to a mammalian GBS toxin receptor comprises (1) simulating andselecting the most probable conformations of a GBS toxin receptor orfragment thereof, (2) designing a chemically modified analog thatsubstantially mimics the energetically most probable three-dimensionalstructure of the GBS toxin receptor or fragment, (3) chemicallysynthesizing the analog, and (4) evaluating the bioactivity of theanalog. Preferably, steps (a) and (b) are performed with the aid of acomputer program.

A preferred method for designing a compound which binds to a mammalianGBS toxin receptor comprises (1) simulating and selecting the mostprobable conformations of a GBS toxin receptor or fragment thereof, (2)deducing most probable binding domains of the receptor or fragment, (3)designing a compound that would form the energetically most probablecomplexes with the receptor or fragment, (4) chemically synthesizing thecompound, and (5) evaluating the bioactivity of the compound.Preferably, steps (a)-(c) are performed with the aid of a computerprogram.

Preferred polypeptides for use in the screening assays described aboveare polypeptides sharing at least about 85% identity, preferably atleast about 95% identity, and most preferably greater than about 99%identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4,or a fragment thereof having GBS toxin receptor activity. Most preferredare polypeptides having an amino acid sequence of SEQ ID NO: 2, 4 OR 8or a fragment thereof having GBS toxin receptor activity.

Methods of Purification

Another aspect of the invention is a method for purifying a compoundthat binds a GBS toxin receptor, for example, natural ligand, otherpolysaccharides, or an antibody specific for the GBS toxin receptor. Themethod comprises providing a polypeptide comprising a mammalian GBStoxin receptor or fragment thereof that binds GBS toxin, contacting thepolypeptide with a sample comprising the compound under conditions thatallow specific binding of the compound to the polypeptide, andseparating the bound compound from the remainder of the sample. Thepolypeptide can be soluble but preferably is immobilized on a substratee.g., on a bead, membrane or on the surface of a cell, preferably astable transfected cell.

Methods of Treatment

GBS toxin receptor polypeptides and antibodies that interfere with GBStoxin binding can be used in a method of treatment of the human oranimal body. For example, such inhibitors of GBS toxin binding can beadministered to a patient to treat or prevent medical conditionsinvolving GBS toxin binding to a GBS toxin receptor, such as, forexample, early onset disease in the neonate.

GBS toxin mimetics or other compounds that bind and/or inhibit GBS toxinreceptor, some of which can be identified by the drug discovery assaysof the invention, can be used in a method of treatment of the human oranimal body or can be used for the manufacture of a medicament for thetreatment or prevention of any of a number of medical conditionsinvolving pathologic and/or hypoxia-driven angiogenesis, such as, forexample, cancerous tumors, chronic inflammatory diseases, scarringduring wound healing or repair of neural injury.

In a preferred embodiment, such a compound exerts its therapeutic effectby binding GBS toxin receptor and evoking an inflammatory response, asdoes GBS toxin. Preferably, such compounds comprise a sulfhydryl,hydroxyl, or amino group displayed so as to be available for bindingcomplement C3.

In another preferred embodiment, the compound is an inhibitor of GBStoxin activity. Preferred inhibitors include, but are not limited to,kinase inhibitors, single chain antibodies specific for the GBS toxinreceptor, and antisense polynucleotides that specifically hybridizeunder high stringency conditions to a GBS toxin receptor nucleic acidsequence, such as that of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:7.

In another preferred embodiment, the compound exerts its therapeuticeffect without evoking an inflammatory response. The compound can beused to deliver a cytotoxic agent to tissue in close proximity to a cellexpressing a GBS toxin receptor, such as, for example, a tumorundergoing angiogenesis. Preferably, the compound is covalently attachedto a cytotoxic agent and can be associated non-covalently with acytotoxic agent, such as, for example, on the external surface of aliposome, micelle, or other lipophilic drug encapsulating structure.Preferred cytotoxic agents include antineoplastic agents commonly knownin the art, such as, for example, mechlorethamine, chlorambucil,cyclophosphamide, melphalan, ifosfamide, and other alkylating agents,methotrexate and other folate antagonists, 6-mercaptopurine and otherpurine antagonists, 5-fluorouracil and other pyrimidine antagonists,cytarabine, ovinblastine, vincustine, and other vincas, etoposide andother podophyllotoxins, doxorubicin, bleomycin, mitomycin, and otherantibiotics, carmustine, lomustine and other nitrosureas, cisplatin,interferon, asparaginase, tamoxifen, flutamide, and taxol. Otherpreferred biologic agents include sense and/or antisense RNA or DNAsequences derived from specific tumor promoter or suppressor genes, suchas, for example, the p53 and TGF gene families, signal transductionprotein family members such as, for example, ras and myc, and growthfactor receptor kinases such as, for example flt2 and flk1, Tai1, Tai2,and neuropholin, and other genes implicated in neoplastic disease andother diseases driven by pathologic angiogenesis.

In another embodiment, GBS toxin receptor polypeptide or fragmentthereof can be administered to a subject as a decoy to reduce the amountof stimulation of the GBS toxin receptor present in afflicted tissues(e.g., tumor tissues), thereby reducing cellular responses leading toproliferation and migration of cells of the afflicted tissues.Preferably, the GBS toxin receptor polypeptide or fragment isadministered in soluble form, even more preferably sans transmembranedomains.

Pharmaceutical Compositions

Polypeptides of the invention that comprise a domain essential for GBStoxin binding that have the desired characteristics for bioavailability,stability and other important parameters of pharmacokinetics in vivo canbe used as a competitive inhibitor of GBS toxin binding for medicalconditions, such as, for example, early onset disease in the neonate, inwhich GBS toxin binding is undesirable. Appropriate polypeptides caninclude fragments having an amino acid sequence corresponding to apartial or full sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or analogsthereof.

Compounds determined by assays using the polypeptides of the inventionto bind and/or GBS toxin receptor and/or induce an inflammatoryresponse, and that have the desired pharmacokinetic characteristics, canbe used as treatments for medical conditions in which GBS toxin bindingcan be therapeutic, such as, for example, medical conditions involvingpathologic or hypoxia-driven angiogenesis or neovascularization.

Pharmaceutical compositions of the invention include a pharmaceuticallyacceptable carrier that may contain a variety of components that providea variety of functions, including regulation of drug concentration,regulation of solubility, chemical stabilization, regulation ofviscosity, absorption enhancement, regulation of pH, and the like. Forexample, in water soluble formulations the pharmaceutical compositionpreferably includes a buffer such as a phosphate buffer, or otherorganic acid salt, preferably at a pH of between about 7 and 8. Othercomponents may include antioxidants, such as ascorbic acid, hydrophilicpolymers, such as, monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, dextrins,chelating agents, such as EDTA, and like components well known to thosein the pharmaceutical sciences, e.g. Remington's Pharmaceutical Science,latest edition (Mack Publishing Company, Easton, Pa.).

An effective amount of an active compound such as a GBS toxin receptorpolypeptide, mimetic or analog, or GBS toxin mimetic or analog forparticular applications depends on several factors, including thechemical nature of the polypeptide, mimetic or analog, the disorderbeing treated, the method of administration, and the like. Preferably,an effective amount will provide a concentration of polypeptide ormimetic of between about 0.0001 to 100 μm at the target GBS toxinreceptor on a cell surface, more preferably less than 10 μm, with lessthan 1 μm being most preferred.

The active compound can be administered to a mammalian host in a varietyof forms, i.e., they may be combined with various pharmaceuticallyacceptable inert carriers in the form of tablets, capsules, lozenges,troches, hard candies, powders, sprays, elixirs, syrups, injectable oreye drop solutions, and the like depending on the chosen route ofadministration, e.g., orally or parenterally. Parenteral administrationin this respect includes administration by the following routes:intravenous, intramuscular, subcutaneous, intraocular, intrasynovial,transepithelial (including transdermal, ophthalmic, sublingual andbuccal), topical (including ophthalmic, dermal, ocular, rectal, nasalinhalation via insufflation and aerosol), and rectal systemic.

The active compound may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, it may be enclosedin hard or soft shell gelatin capsules, compressed into tablets, orincorporated directly with the food of the diet. For oral therapeuticadministration, the active compound may be incorporated with excipientand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at lease 0.1% of activecompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2% to about 6%of the weight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 1 and 1000 mg of active compound.

Tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as polyvinylpyrrolidone, gum tragacanth,acacia, sucrose, corn starch or gelatin; an excipient such as calciumphosphate, sodium citrate and calcium carbonate; a disintegrating agentsuch as corn starch, potato starch, tapioca starch, certain complexsilicates, alginic acid and the like; a lubricant such as sodium laurylsulfate, talc and magnesium stearate; a sweetening agent such assucrose, lactose or saccharin; or a flavoring agent such as peppermint,oil of wintergreen or cherry flavoring. Solid compositions of a similartype are also employed as fillers in soft and hard-filled gelatincapsules; preferred materials in this connection also include lactose ormilk sugar as well as high molecular weight polyethylene glycols. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, flavoring such as cherry or orange flavor,emulsifying agents and/or suspending agents, as well as such diluents aswater, ethanol, propylene glycol, glycerin and various combinationsthereof. Of course, any material used in preparing any dosage unit formshould be pharmaceutically pure and substantially non-toxic in theamounts employed. In addition, the active compound may be incorporatedinto sustained-release preparations and formulations.

The active compound may also be administered parenterally orintraperitoneally. For purposes of parenteral administration, solutionsin sesame or peanut oil or in aqueous propylene glycol can be employed,as well as sterile aqueous solutions of the corresponding water-soluble,alkali metal or alkaline-earth metal salts previously enumerated. Suchaqueous solutions should be suitably buffered, if necessary, and theliquid diluent first rendered isotonic with sufficient saline orglucose. Solutions of the active compound as a free base or apharmacologically acceptable salt can be prepared in water suitablymixed with a surfactant such as hydroxypropylcellulose. A dispersion canalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal injection purposes. In this connection, the sterileaqueous media employed are all readily obtainable by standard techniqueswell-known to those skilled in the art.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of a dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and the freeze drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from the previously sterile-filtered solutionthereof.

For purposes of topical administration, dilute sterile, aqueoussolutions (usually in about 0.1% to 5% concentration), otherwise similarto the above parenteral solutions, are prepared in containers suitablefor drop-wise administration to the eye. The compounds of this inventionmay be administered to a mammal alone or in combination withpharmaceutically acceptable carriers. As noted above, the relativeproportions of active ingredient and carrier are determined by thesolubility and chemical nature of the compound, chosen route ofadministration, the particular compound chosen and the physiologicalcharacteristics of the particular patient under treatment.

Kits

Yet another aspect of the invention is a kit for use in carrying out anyof the above methods. A preferred embodiment is a kit comprising a GBStoxin receptor or fragment thereof. Preferably, the receptor or fragmentis immobilized. A preferred kit can be used for identifying a compoundthat binds to GBS toxin receptor, and comprises at least one cell thatexpresses GBS toxin receptor.

Another embodiment is a kit for monitoring tumor growth or metastasis,comprising a reagent for detecting expression of a GBS toxin receptor.Examples of such reagents include, but are not limited to,polynucleotide probes that hybridize to a GBS toxin receptor nucleicacid sequence and compounds that bind to a GBS toxin receptor, such as,for example, an antibody that specifically recognizes GBS toxinreceptor, a GBS toxin, a GBS toxin mimetic, or other compoundsidentified by the screening methods described above.

A third embodiment is a kit for purifying a compound that binds a GBStoxin receptor, comprising a GBS toxin receptor or fragment thereof thatbinds the compound. Preferred compounds include GBS toxin, GBS toxinmimetics, antibodies that specifically bind GBS toxin receptor, andother compounds identified by the screening methods described above.

Additional kit components can include, but are not limited to,additional reagents required for detection, a reference standard(s),instructions for use, and the like. Suitable reference standards includepositive controls, negative controls, photographs of such controls,tabulated or graphed data of such controls, and the like. The kits mayfurther comprise instructions for carrying out the methods describedabove, preferably printed instructions.

Examples Example 1 Cloning Sheep GBS Toxin Receptor

Primary Culture of Sheep Lung Endothelial Cells

Small pieces of primary lung tissues from a 7-week old sheep are cutinto small pieces in Hank's balanced salt solution (HBSS) containing 10mM HEPES buffer (Life Technology), 1% penicillin/streptomycin and 0.1%gentamycin, and are cultured in sheep lung complete medium (LifeTechnology) at 37° C. After one week of the culture, clones of sheeplung endothelial cells are identified by Cobblestone morphology andharvested into 24-well tissue culture plates (Falcon) using cloningrings. When the cells are confluent, they are detached by pancreatin andtransferred to a 60-mm tissue culture Petri dish or a T-25 tissueculture flask (Falcon). When they are confluent again, they are splitand cultured into a few 100-mm tissue culture plates (Falcon). Eachsplit is considered to be one passage. The same procedure is repeateduntil enough cells (˜10⁸) are obtained for isolation of mRNA.

Isolation of mRNA and Construction of cDNA Library

Poly(A)+RNA is isolated from 9.2×10⁷ sheep lung endothelial cells(passage 8 and 9) by a standard method (Pharmacia). A total of 16 μgpoly(A)⁺ RNA is acceptable amount obtained. 2.5 μg mRNA can be used toconstruct a cDNA library. Poly(A)⁺ RNA is oligo(dT)-primed (with NotIrestriction site) and converted into double-stranded cDNA. After addinga BstXI/EcoRI adaptor, the cDNA is unidirectionally cloned into theBstXI and NotI sites of pCDNA3.1(+) (Invitrogen). E. coli Top10F′(Invitrogen) is used as a host strain for amplification. 5.38×10⁶primary clones are an acceptable number generated. The library isamplified by plating cells onto fifty large LB agar plates containingampicillin (100 μg/ml). The plates are scraped and aliquoted so thateach aliquot represents 10 plates. DNA is purified by Qiagen Max columns(Qiagen).

Screening of cDNA Library for a Gene Encoding GBS Toxin Receptor

To screen a cDNA library for a gene encoding GBS toxin receptor gene, aunique colorimetric method is used. Five μg plasmid DNA from each poolof cDNA library is used to transfect COS7 cells. The transfected cellsare cultured in four to eight 96-well tissue culture plates (Falcon) fortransient expression. Each well contains about 20,000 transfected cellsin DMEM medium (Life Technology). COS7 cells transfected withpCDNA3.1(+) are used as a control. After 3 days expression, the mediumis carefully removed. Each well is rinsed 3 times with HPSS buffercontaining Mg²⁺ and Ca²⁺ (wash buffer) (Life Technology).

The cells are then incubated with biotinylated toxin (50 μl per well; 1to 1.5 μg/ml) at room temperature for 1 h. After the hour incubation,the biotinylated toxin is discarded and the wells are rinsed 3 timeswith the wash buffer. The cells are incubated with streptavidin-β-galsolution and each well is rinsed 3 times with the wash buffer. The cellsare then incubated with PNPG (50 μl per well; 1 mg/ml in substratebuffer) at 37° C. Absorbance at 405 nm is measured by an ELISA reader at1 and 20 h, respectively. The cells which give the highest OD areharvested. Plasmid DNA is isolated by Hirt extraction. Plasmid DNA isamplified in E. coli to have enough DNA for the next transfection(enrichment).

Enrichment is done 8 times by this colorimetric method. The number ofthe transfected cells loaded into each well is gradually decreased inthe last few enrichments and untransfected cells are added to each wellto give a total number of 20,000 cells per well for the cells to beconfluent and to reduce background after 3 days' expression. At the lastenrichment, each well has only 1 to 10 transfected cells. Cells givingthe highest OD are harvested. DNA is isolated and amplified in E. coli.

A number of isolated clones are individually assayed by thiscolorimetric method. The clones which showed higher binding to CM101 aresequenced.

Sequence Analysis

DNA sequence analysis of clone pFU102, which has a 2.1 kb insert,revealed a sequence encoding a partial integral glycoprotein. N-terminalsequence was obtained by 5′RACE method (Life Technology) and afull-length gene is designated as SP55. Triple ligation yielded pCD55,which contains an entire coding region of SP55.

mRNA for the SP55 has 2844 nucleotides, encoding a protein of 495 aminoacids with a predicated mass of 55 KDa, SP55. Analysis by the method ofKlein et al. (Klein et al., Biochim Biophys Acta, 815:468-476 (1985))classifies SP55 as an integral protein with seven transmembranesegments. SP55 has both N-glycosylation and kinase phosphorylationsites. A Swiss-Prot, search of SP55 did not reveal any high homology toknown human proteins. However, SP55 has some identity (˜30%) to renalsodium-dependent phosphate transporters from human, rabbit, mouse andrat. In addition, SP55 has some identity (˜30 to 39%) to hypotheticalproteins (HYP50 and HYP63) from C. elegans.

Example 2 Cloning Human GBS Toxin Receptor

The sheep GBS toxin receptor sequence shares about 37% identity withHYP50 and about 33% identity to HYP63, two hypothetical proteins from C.elegans. In the regions corresponding to amino acid residues 180-186 and443-449 of SEQ ID No. 2, five amino acids within a seven amino acidstretch are absolutely conserved among the three proteins.

A first degenerate oligonucleotide, CMR3-S:5′-CGGGATCCCGCCNGCNATGCAYRSHRTSTGG-3′(SEQ ID No. 5), was designed toinclude all possible codons encoding the amino acid sequences of SP55,HYP50, and HYP63 in the 180-186 region. A second degenerateoligonucleotide, CMR4-AS2: 5′-GGAATTCCDGGDGCRATKTCNARRTRRTT-3′ (SEQ IDNo. 6), was designed to include the complementary sequences of allpossible codons encoding the amino acid sequences of SP55, HYP50, andHYP63 in the 443-449 region.

Polymerase chain reaction (PCR) was conducted using theseoligonucleotides and a human embryo lung cDNA library as a template. Thereaction yielded three overlapping sequences approximately 400 by insize, which encompass part of the nucleic acid sequence of SEQ ID No. 3.These sequences were then used as probes to clone the remainder of thegene, referred to herein and HP59 (SEQ ID NO: 7).

Example 3 Preparation of Antibodies Against GBS Toxin Receptor

Rabbits are immunized with the synthetic peptides shown in Table 8. A 1mg/ml solution of peptide plus KLH in 0.01M phosphate buffer isprepared. For the first immunization, 200 μg of peptide plus KLH (200μl) and an equal volume of Freunds complete adjuvant, emulsified wellbefore injection, is injected into 3-4 spots on the dorsal surface aboutthe neck and shoulders of a rabbit. After two weeks, the secondimmunization (boost) is given at the same concentration of immunogen,but emulsified in Freunds incomplete adjuvant. The boost is delivered inthe same region of the body. After another two weeks, blood is collectedand assayed by ELISA for response against the peptide without KLH.Further boosts are given to improve antibody titer, if necessary.

TABLE 8 Immunogenic Peptides Pep- tide Amino Acid Sequence Size SEQ IDRef. p56a APSDGEEGSDRTPLLQRAPRAEPAPVC 27 residues 8-35 aa of SEQ ID NO:4 p55a LAPSDGEEGSDRTPL 15 residues 7-22 aa of SEQ ID NO: 4 p57aNTTAKDNRTSYECA 14 residues aa 71-84 of SEQ ID NO: 4

Peptide p55 is a fragment of an extracellular domain of GBS toxinreceptor. Peptide p57a is a fragment of an intracellular domain of GBStoxin receptor. Animals immunized with these peptides produce polyclonalantibodies Pab55 and Pab57, respectively.

Example 4 Detection of GBS Toxin Receptor Expression in Tumor Cells

This example shows that GBS toxin receptor can be detected in tumorcells. Immunohistochemistry is performed on paired human and mousetissues of normal or tumor origin, using rabbit polyclonal antibodiesPab 55 and Pab 57.

Mouse and human tumor tissues are fixed in 10% neutral formalin. Thetissues are then dehydrated, paraffin embedded and 10-20×8-micronsections are cut for immunohistochemical staining.

Immunohistochemical analysis is performed with the automated VentanaImmunohistochemical Stainer according to the manufacturer's suggestedprotocol (Ventana, Tucson, Ariz.). Sections are deparaffinated withxylene. The prepared sections are then treated with 1% hydrogen peroxideprepared in 30% aqueous methanol for 20 minutes at room temperature toquench endogenous peroxidase activity. The slides are then washed withPBS, blocked with 5% BSA and 5% goat serum in PBS, washed again and thenincubated for 30 minutes at 37° C. with the appropriate diluted (1:100)antibody. Horseradish peroxidase-labeled goat anti-rabbit IgG is used asa secondary antibody. For visualization, the sections are incubated withDAB/H₂O₂. The sections are finally incubated with a copper enhancer(Ventana) for 4 minutes, washed, counterstained with hematoxylin, andmounted in toluene-minus mounting medium. Photographic documentation isperformed and images are stored for later review and analysis. Theresults are summarized in Table 9. The numbers refer to glass slides.

TABLE 9 Immunohistochemistry of tumor and normal tissues AntibodyMagnification Signal Human tissues: 1. Ovary tumor (95-02VO16) highgrade papillary carcinoma Pab 55 400x + 2. Ovary tumor (95-02VO16) highgrade papillary carcinoma Pab 55 400x + 3. Normal ovary (96-08ZO08)control tissue Pab 55 400x − 4. Ovary tumor (95-02VO16) high gradepapillary carcinoma Pab 57 400x + 5. Ovary tumor (95-02VO16) high gradepapillary carcinoma Pab 57 400x + 6. Ovary tumor (95-02VO16) high gradepapillary carcinoma Pab 57 400x + 7. Normal ovary 96-08ZO08) control Pab57 400x − 8. Colon cancer 95-14664) poorly diff. Adenocarcinoma Pab 55400x + 9. Normal colon 9708VO08) control Pab 55 400x − 10. Colon cancer95-14664) poorly diff. Adenocarcinoma Pab 57 400x + 11. Colon cancer95-14664) poorly diff. Adenocarcinoma Pab 57 400x + 12. Normal colon9708VO08) control Pab 57 400x − 13. Female breast cancer (97-IOV03a)Invasive mammary carcinoma Pab 55 400x + 14. Male breast cancer (nocode) mammary carcinoma Pab 55 400x + 15. Normal female breast97-12VO20-3) control Pab 55 400x − 16. Female breast cancer 97-IOV03a)Invasive mammary carcinoma Pab 57 400x + 17. Male breast cancer (nocode) mammary carcinoma Pab 57 400x + 18. Normal female breast(97-12VO20-3) control Pab 57 400x − 19. Lung cancer (97-1OV022-5) poorlydiff. NOJ-small cell carcinoma Pab 55 400x + 20. Normal lung (98-01VO11)control Pab 55 − 21. Lung cancer (97-10VO22-5) poorly diff. NOJ-smallcell carcinoma Pab 57 400x + 22. Lung cancer (97-10VO22-5) poorly diff.NOJ-small cell carcinoma Pab 57 400x + 23. Normal lung (98-01VO11)control Pab 57 − Mouse Tissues: 24. Madison Lung Tumor (MLT) untreatedwith CM 101 Pab 55 + 25. MLT untreated with CM 101 Pab 55 + 26. Normalmouse lung Pab 55 − 27. MLT untreated with CM 101 Pab 57 + 28. Normalmouse lung Pab 57 − (diff. = differentiated)

The Pab 55 antibody stains the cells lining a blood vessel in a humanovary cancer tissue section, but such staining is not apparent in cellsof normal human ovary tissue (see FIGS. 2A and 2B, respectively).Similar results are obtained with the Pab 57 antibody (see FIGS. 3A and3B). As shown in the above table and in FIGS. 2A-3B, antibodies raisedto GBS toxin receptor fragments specifically bound to tumor tissues butnot normal tissues, suggesting that GBS toxin receptor is expressed intumor cells but not normal cells.

Example 5 Detection of GBS Toxin Receptor Expression in Mice Afflictedwith Rheumatoid Arthritis

This example shows that GBS toxin receptor can be detected in cells froma mammalian model for rheumatoid arthritis (RA). Mice withcollagen-induced arthritis were treated with CM101 or carrier. CM101reversed the inflammatory damage and inhibited pannus formation. Mouse#8 and #15, which were treated with CM101, and two control mice (nottreated with CM101) were sacrificed for immunohistochemistry.

TABLE 10 Immunohistochemistry of Rheumatoid Arthritic Mice 29. No CM 101Pab 55 + 30. MOUSE 8-5′ (vessel) Pab 55 + 31. No CM 101 Pab 57 + 32.MOUSE 15-5′ (vessel) Pab 57 + 33. MOUSE 8-5′ (between joint) Pab 57 +34. MOUSE 15-5′ Pab 57 + 35. No CM 101 (marrow) Pab 57 + 36. MOUSE 15-5′(marrow) Pab 57 +

As shown above Pab55 and Pab57 specifically bound to pathologicneovasculature in the pannus, suggesting that GBS toxin receptor isexpressed in mice afflicted with rheumatoid arthritis. No binding ofCM101 was observed in the normal neovasculature in the growth plate ofthe joints of the arthritic mice.

Example 6 Targeted Delivery of a Chimeric Compound to Tissues ExpressingGBS Toxin Receptor

This example shows the targeted delivery of a chimeric compound totissues expressing GBS toxin. The chimeric compound is a CM101-biotinconjugate. Mice with Madison Lung Tumors (MLT) are infused intravenously(i.v.) with biotinylated CM 101.

CM101 has been reacted with hydrazinylated biotin to form the biotinhydrazone at the reducing end of the polysaccharide CM101. Briefly, 25micrograms of lyophilized CM101 is dissolved in 250 μl labeling bufferat 100 mM sodium acetate, 0.02% sodium azide. Aqueous meta-periodate(125 μl of 30 mM) is added and the oxidation is allowed to proceed inthe dark for 30 minutes at room temperature. The reaction is terminatedby adding 80 mM Na₂SO₃ to the solution. The resultant aldehydes arereacted with 125 μl of 5 mM NHS-LC-Biotin (MW 556.58) for a 1 hourincubation at room temperature to form biotinylated CM101. Excess biotinis removed by dialysis against 1 liter of PBS at 4° C. four times. Theproduct is purified by gel filtration on an Ultrahydrogel 1000 HPLC,lyophilized and stored at −70° C. until use.

Tissues are recovered 5 min post infusion with CM101 and subjected toimmunohistochemistry. Tumor and normal mouse tissue sections areanalyzed for CM 101 binding by both mouse anti-CM101 mAb (7A3), followedby secondary mAb-HRP conjugate (referred to in FIG. 4B as MLTCM101-Biot.5′+McAb), or with avidin (which specifically binds biotin)conjugated with HRP (referred to in FIG. 4A as MLTCM101-Biot.5′+Strep.HRP).

FIGS. 4A-4C depict different sections taken from the same tumor andinclude a longitudinal view of the same blood vessel approximately inthe center of the figures. The dark staining in FIG. 4A shows thelocalization of the biotin component in the cells lining the bloodvessel. Similarly, FIG. 4B depicts the localization of the CM101component in the cells lining the blood vessel. FIG. 4C is a negativecontrol that was not exposed to CM101. The analysis clearly shows that7A3 and avidin bind to the same blood vessels in tumor tissue. Thus,biotin has been delivered to the blood vessel of the tumor tissue byvirtue of its physical association with a compound (CM101) that bindsthe GBS toxin receptor.

These studies show that chimeric compounds can be delivered to tissuesundergoing pathologic and/or hypoxia-driven angiogenesis orneovascularization. As part of a chimeric compound, cytotoxic moleculescan be directed to such tissues, e.g., tumor tissue. The cytotoxicmolecule can be coupled directly to a molecule that binds GBS toxinreceptor, e.g., GBS toxin. Alternatively, the molecule that binds GBStoxin receptor can be coupled to biotin and the cytotoxic molecule canbe coupled to avidin.

Example 7 Enhanced Sensitivity to GBS-Toxin-Dependent Cytotoxicity ofCells Expressing GBS Toxin Receptor

This example shows the enhanced sensitivity to GBS-toxin-dependentcytotoxicity of cells transfected with the GBS toxin receptor, relativeto control cells. Without being bound to a particular theory, theinventors believe that complement binds GBS toxin bound to the GBS toxinreceptor on a cell, thereby targeting the cell for killing by whiteblood cells (WBC).

Human bladder carcinoma cells (ECV cells), are stable transfected withthe human GBS toxin receptor gene. The resultant cell line is ECV711.Cells stable transfected with vector alone as referred to as V23. ECV711 and V23 are seeded in 96-well plates at 5,000 cells/well.

White blood cells are collected from healthy human donors as follows.Blood is collected by standard phlebotomy procedures into heparinizedtubes (30 U/ml) and centrifuged at 2000 rpm for 20 min. The interface iscarefully transferred to a new tube and washed twice by centrifugationwith medium (RPMI-1640). Cells are resuspended in RPMI-1640 supplementedwith 5% fetal bovine serum (FBS) and Interferon-gamma (IFN) at 100 U/ml,and incubated overnight in a 37° C., 5% CO₂ incubator. The cells arethen resuspended in fresh medium with 5% FBS.

5,000 cells of the WBC preparation are added to each well containing thetransfected cells. CM101 is added to a final concentration of 1 μg/ml tothe wells together with human serum from matching human donors. Thecells are incubated 6 hours at 37° C.

Cytotoxicity is assayed by measuring lactate dehydrogenase (LDH) usingthe Promega's CytoTox 96 Non-Radioactive Assay kit (Nachlas et al.(1960) Anal. Biochem 1, 317; Korzeniewski et al. (1983) J. Immunol.Methods 64, 313; Decker et al. J. Immunol. Methods 115, 61; Brander etal. (1993) Eur. J. Immunology 23, 3217; Behl et al. (1994) Cell 77, 817;Lappalainen et al. (1994) Pharm. Research 11, 1127; Allen et al. (1994)Promega Notes 45, 7; Sinensky et al. (1995) Toxicol. Letters 75, 02;Moravec (1994) Promega Notes 45, 11). Percent cytotoxicity is calculatedas recommended by the manufacturer's instructions. The results are shownin Table 11.

TABLE 11 Cytotoxicity ECV 711 V 23 WBC, IFN, C3,  29.1%  27.5% −CM101WBC, IFN, C3, 40.45% 22.46% +CM101

There is an increase in cytotoxicity of 39% when the ECV 711 cells areincubated with CM101, WBC and human serum (source of C3) compared tocells incubated without CM101. Control cells transfected with vectoralone, V23, do not show a CM101 dependent increase in cytotoxicity.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. An isolated polynucleotide at least 10 bases in length comprising anucleic acid sequence encoding, a mammalian receptor for group Bβ-hemolytic Streptoccocus toxin (GBS toxin receptor), or a polypeptidefragment thereof. 2-81. (canceled)